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V 


ENZYMES 

AND  THEIR  APPLICATIONS. 


BY 

DR.    JEAN    EFFRONT, 

PROFESSOR    IN    THE    NEW    UNIVERSITY    IN    BRUSSELS    AND 
DIRECTOR    OF    THE    FERMENTATION    INSTITUTE. 


ENGLISH  TRANSLATION 

BY 

SAMUEL    C.    PRESCOTT,    S.B., 

INSTRUCTOR    IN    INDUSTRIAL    BIOLOGY, 
MASSACHUSETTS    INSTITUTE    OF    TECHNOLOGY,    BOSTON. 


Volume  I. 

THE    ENZYMES    OF    THE    CARBOHYDRATES. 
THE  OXIDASES. 


FIRST    EDITION. 
FIRST    THOUSAND. 


NEW  YORK: 

JOHN   WILEY    &    SONS. 

London:  CHAPMAN  &  HALL,  Limited. 

1902. 


Copyright,  1902, 

BY 

JOHN  WILEY  &  SONS. 


ROBERT    DRUMMOND      PRINTER      NEW   VORK. 


AUTHOR'S  PREFACE. 


The  study  of  chemical  ferments  affords  the  double  advan- 
tage of  presenting  a  broad  scientific  interest  and  having  at 
the  same  time'  numerous  industrial  applications.  The  phe- 
nomena of  assimilation  and  respiration  which  take  place  in 
the  interior  of  the  living  cell  are  in  direct  relation  to  the 
diastatic  secretions,  the  study  of  which  is  consequently  of  as 
great  importance  to  physiologists  as  to  botanists  and  bac- 
teriologists. A  knowledge  of  the  reactions  caused  by  the 
diastases  is  also  of  first  importance  to  chemists,  for  whom 
these  physiological  agents  may  become  reagents  of  an  ex- 
ceptional sensitiveness.  The  science  of  chemical  ferments 
comprises  also  the  knowledge  of  certain  microbial  poisons, 
which,  by  their  properties,  are  singularly  allied  to  ordinary 
diastases.  To  study  these  poisons  from  the  point  of  view  of 
their  diffusion,  conservation,  and  destruction  in  the  organ- 
ism, one  must  also  possess  an  accurate  knowledge  of  en- 
zymes. Finally,  a  whole  class  of  soluble  ferments  have  found, 
at  the  present  time,  industrial  application,  and  undoubtedly 
the  future  will  add  many  others ;  here,  then,  is  a  further  in- 
terest which  attaches  to  the  study  of  enzymes. 

The  present  work,  which  is  a  summary  of  the  course 
given  at  the  Institute  of  Fermentations  of  the  new  Univer- 
sity of  Brussels,  is  designed  both  for  persons  who  give  them- 
selves up  to  purely  scientific  studies  and  for  those  who  are 
occupied  particularly  in  fermentation  industries.  So,  while 
reserving  the  largest  place  for  theoretical  questions,  we  have 


347505 


iv  AUTHOR'S  PREFACE. 

not  neglected  the  practical  results.  Our  work  is  divided 
into  two  parts.  In  the  first,  which  constitutes  the  present 
volume,  we  deal  with  the  enzymes  of  carbohydrates  and  with 
the  oxidases,  as  well  as  with  their  industrial  applications.  In 
the  second  part,  now  in  preparation,  we  shall  study  the  pro- 
teolytic enzymes  and  the  toxins. 

We  have  personally  verified  the  greater  part  of  the  ex- 
perimental data  which  this  first  volume  contains,  in  which  the 
reader  will  find  a  certain  number  of  hitherto  unpublished  ex- 
periments, methods  of  preparation,  methods  of  analysis,  and 
technical  processes. 

Brussels,  1898. 


TRANSLATOR'S    NOTE. 


Up  to  the  present  time  very  few  works  upon  Enzymes 
have  appeared  in  our  language.  In  the  translation  of  Profes- 
sor Effront's  book  I  have  been  actuated  by  the  desire  to 
make  available  in  English  the  valuable  material  contained  in 
the  original  French  edition.  I  have  endeavored  to  reproduce 
the  author's  ideas  with  clearness  and  simplicity,  without  at 
the  same  time  deviating  too  much  from  his  own  form  of  ex- 
pression. I  have  made  no  additions  or  changes,  and  the 
book  is  therefore  presented  in  a  form  as  nearly  as  possible 
like  that  in  which  it  came  from  the  pen  of  the  author. 

I  wish  to  acknowledge  my  indebtedness  to  my  friend,  Mr. 
Percy  G.  Stiles  of  Johns  Hopkins  University,  for  much  as- 
sistance in  proof  revision. 

Samuel  C.  Prescott. 

Massachusetts  Institute  of  Technology, 
December,  1901. 


TABLE  OF  CONTENTS. 


PAGE 

Preface "*• 

CHAPTER  I. 

GENERAL   REMARKS. 

Synthetical  and  analytical  work  of  the  living  cell.— Synchronism 
of  the  two  phenomena. — Difference  between  chemical  and  physio- 
logical activity. — Chemical  agents  and  physiological  agents. — In- 
tervention of  vital  energy.— Necessity  of  studying  the  physical 
and  chemical  conditions  of  the  medium. — Definition  of  enzymes. 
■ — Their  part  in  assimilation  and  disassimilation. — Enzymes  as 
producers  of  heat I 

CHAPTER  II. 

GENERAL   PROPERTIES. 

History  of  the  knowledge  of  enzymes. — Works  of  Reaumur  and 
Spallanzani,Kirchoff,  Dubrunfaut  and  Payen. — General  properties 
of  diastases. — Means  of  distinguishing  a  diastatic  action  from  a 
purely  chemical  action. — Test  with  tincture  of  guaiacum. — Law  of 
proportionality  in  diastatic  action. — Means  of  distinguishing  the 
work  of  organized  ferments  from  diastatic  action. — Means  of 
isolating  the  diastase  from  the  medium  which  contains  it. — Chem- 
ical composition  of  enzymes. — Zymogenesis. — Method  of  action 
of  diastases ifc 

CHAPTER  III. 

MANNER   OF   ACTION    OF    DIASTASES. 

Manner  of  action  of  diastases. — Different  opinions  on  this  subject. — 
The     diastatic     property     and     the     diastase     itself. — Works     of 

vii 


Tin  TABLE  OF  CONTENTS. 


Bunzen,  Hiifner,  Naegeli,  Wittich  and  Fick,  de  Jager,  Arthus. — 
Analogy  between  organized  ferments  and  soluble  ferments. — 
Hypothesis  of  Armand  Gautier  on  the  nature  of  enzymes. 27 


CHAPTER  IV. 

INDIVIDUALITY   OF   ENZYMES. 

Difficulties  encountered  in  proving  the  individuality  of  enzymes. — In- 
fluence of  the  manner  of  nutrition  of  the  cells  on  the  nature  of  the 
enzymes  they  secrete. — Direct  proofs  of  the  individuality  of  en- 
zymes.— Relation  between  the  diastases;  the  chemical  constitution 
arid  the  structure  of  the  bodies  on  which  they  act. — Nomenclature 
of   enzymes. — Classification 4<jn 


CHAPTER  V. 


Extraction  of  sucrase  from  yeast. — Secretion  by  Aspergillus  niger. — 
Preparation  of  sucrase  in  the  dry  state. — Influence  of  the  quan- 
tity and  of  time. — Influence  of  temperature. — Difference  between 
the  properties  of  sucrases  of  different  origin. — Effect  of  acidity 
or  alkalinity  of  the  medium. — Action  of  oxygen  and  of  light. — 
Action  of  chemical  substances. — Mode  of  secretion  of  sucrase  in 
the  cells. — Measurement  of  sucrase. — Fernbach's  method. — Ef- 
front's  method 50 


CHAPTER   VI. 

SUCRASE    (CONTINUED). 

Retarding  factors,  and  their  explanation. — Deterioration  and  altera- 
tion of  sucrase. — Experiments  of  Effront  on  the  influence  exerted 
by  the  invert-sugar  in  the  medium  in  which  inversion  is  pro- 
duced.— Hypothesis  of  O'Sullivan  and  Tompson. — Arguments 
for  and  against  this  hypothesis.  Theory  of  Effront  on  the  decom- 
position of  cane-sugar,  and  experiments  on  the  manner  of  action 
of  acids  in  the  inversion  of  saccharose 75. 

CHAPTER  VII. 

FERMENTATION    OF    MOLASSES. 

Industrial  applications  of  sucrase. — Fermentation  of  molasses 88.' 


TABLE  OF  CONTENTS. 
CHAPTER  VIII. 


Presence  of  amylase  in  vegetable  and  animal  cells. — Preparation. — 
Cohnheim's  method. — Lintner's  method. — Effront's  method. — 
Wroblewsky's  method. — Properties. — Influence  of  quantity,  time, 
and  temperature. — Influence  of  chemical  agents:  acids,  alkalies, 
salts. — Substances  hastening  diastatic  action ioo 

CHAPTER  IX. 

CHEMICAL  WORK   OF   AMYLASE. 

Chemical  work  of  amylase. — Theories  of  Payen  and  Musculus. — 
Existence  of  different  dextrins. — Theory  of  Duclaux  on  the  na- 
ture of  different  dextrins. — Preservation  of  diastases  during  sac- 
charification. — Experiments  of  Effront 121 

CHAPTER  X. 

AMYLASE    OF    DIFFERENT    SOURCES. 

Different  amylases. — Ptyalin. — Diastase  of  raw  grains  and  diastase  of 
germinated  grains. — Action  of  translocation  diastase  on  starch. — 
Reichler's  diastase. — Mode  of  action  of  diastase  brought  to  a  tem- 
perature of  70°. — Conditions  of  secretion  of  amylase. — Quantita- 
tive analysis  of  amylase. — Comparative  value. — Absolute  value. — 
Methods  of  Effront 132 

CHAPTER  XI. 

INDUSTRIAL  APPLICATIONS  OF  AMYLASE. 

Malting. — Chemical  transformations  which  accompany  germination. — 

Methods  of  malting,  sorting,  steeping,  germination,  brewing 147 

CHAPTER  XII. 

R&LE  OF  AMYLASE  IN  THE  BREWERY I55 

CHAPTER  XIII. 

MANUFACTURE  OF  MALTOSE l6l 

CHAPTER  XIV. 

PANARY    FERMENTATION. 

Of  Dumas  theory  of  panary  fermentation. — Cerealine  of  Mege- 
Mouries. — Role  of  bacteria  in  panary  fermentation. — Origin  of 
sugar  in  flour 168 


X  TABLE   OF  CONTENTS. 

CHAPTER  XV. 
r6le  of  amylase  in  the  distillery. 

PAGS 

Treatment  of  grains  by  acid  and  by  malt. — Influence  of  heating  on 
saccharification. — Choice  of  temperatures  of  saccharification. — 
Principal  and  secondary  saccharification. — Experiments  of  Ef- 
front  on  the  alteration  of  diastases  during  saccharification. — The 
infusion  process. — Alteration  of  diastases  during  the  successive 
stages  of  the  work. — Control  of  the  work  in  the  distillery 175 

CHAPTER  XVI. 

QUANTITATIVE   STUDY   OF   MALT. 

Determination  of  the  diastatic  power  of  malt  and  mashes  according 
to  the  methods  of  Effront. — Determination  of  saccharifying  and 
liquefying  powers. — Determination  of  the  diastatic  power  of  sweet 
and  fermented  mashes 195 

CHAPTER  XVII. 

MALTASE. 

Glucase  of  Cusenier. — Maltase  of  yeast. — Properties. — Differences 
between  the  optimum  temperatures  of  the  different  glucases. — 
Maltase  of  moulds. — Mode  of  action  on  starch. — Processes  of 
secretion. — Influence  of  nitrogenous  food. — Different  amylo-malt- 
ases  of  Laborde 208 

CHAPTER  XVIII. 

INDUSTRIAL   APPLICATIONS   OF   MALTASE. 

Cerealose   220 

CHAPTER  XIX. 

INDUSTRIAL    APPLICATIONS    OF    MALTASE. 

Japanese  and  Chinese  yeasts  in  general. — Manufacture  of  Japanese 
yeast. — Preparation  of  "  koji." — Changes  produced  in  the  rice. — 
Composition  of  "koji." — Action  of  salts. — Manufacture  of  "moto" 
leaven. — Manufacture  of  the  beer  "  sake." — Composition  of 
"  moto."  —  Composition  of  "  sake."  —  Manufacture  of  Chinese 
yeast. — Properties  of  Chinese  yeast. — Diastase  of  Chinese  yeast. — 
Influence  of  temperature  and  chemical  agents. — Native  distilleries 
of  the  far  East. — Utilization  of  Oriental  processes  in  the  distil- 
leries of  western  countries. — Works  of  Takamine,  Collette,  and 
Boidin 223 


TABLE   OF  CONTENTS.  xi 

CHAPTER  XX. 

ENZYMES    OF    CARBOHYDRATES. 

PAGE 

Trehalase,  lactase,  inulase,  pectase,  cytase,  caroubinase 246 

CHAPTER  XXI. 

FERMENTS  OF  GLYCERIDES  AND  GLUCOSIDES. 

Saponifying  ferments. — Ferments  of  glycerides. — Serolipase  and  pan- 
creatolipase. — Measurement  of  lipase. — Influence  of  temperature 
and  alkalinity  of  the  medium. — Differences  between  the  lipases 
of  different  origins. — Ferments  of  glucosides. — Myrosin,  emulsin, 
rhamnase,  erythrozyme,  betulase 262 

CHAPTER  XXII. 

ZYMASE. 

Zymase  or  alcoholic  diastase. — Preparation  of  the  extract  of  yeast  and 
its  properties. — Determination  of  the  fermenting  power  of 
zymase. — Chemical  and  physical  conditions  of  the  action  of 
zymase. — Experiments  of  Effront  on  intracellular  fermentation. — 
Industrial  applications  of  zymase 276 

CHAPTER  XXIII. 

OXIDASES. 

Presence  of  oxidases  in  vegetable  and  animal  cells. — General  proper- 
ties.— Laccase. — Tyrosinase. — Influence  of  the  medium. — Action 
of  oxidases  on  phenols  insoluble  in  water. — "Breaking"  of 
wines. — CEnoxidase. — Oxidin. — Olease 292 


THE   ENZYMES  AND  THEIR  APPLICATIONS. 


CHAPTER  I. 
GENERAL  REMARKS. 

Synthetical  and  analytical  work  of  the  living  cell. — Synchronism  of  the 
two  phenomena. — Difference  between  the  chemical  work  and  the  phy- 
siological work. — Chemical  agents  and  physiological  agents. — Inter- 
vention of  vital  energy. — Necessity  of  studying  the  physical  and  chem- 
ical conditions  of  the  media. — Definition  of  enzymes. — Their  part  in 
assimilation  and  disassimilation. — Enzymes  as  producers  of  heat. 

The  activity  of  living-  cells  gives  rise  to  a  series  of  chem- 
ical reactions  very  complex  and  various.  A  limited  observa- 
tion of  phenomena  will  show  that,  aside  from  purely  syntheti- 
cal activity,  the  cell  always  carries  on  analytical  work :  in 
other  words,  that  the  organic  substance,  in  the  presence  of 
the  living  cell,  is  built  up  and  broken  down.  The  synthetical 
activity  is  more  apparent  in  the  transformation  of  substances 
which  are  not  very  complicated,  which,  submitted  to  the  ac- 
tion of  living  cells,  become  hydrated,  oxidized  and  trans- 
formed into  complex  chemical  compounds.  In  the  trans- 
formation of  complex  substances,  on  the  contrary,  it  is  the 
analytical  work  which  predominates,  and  the  complex  sub- 
stance is  reduced  to  more  and  more  simple  bodies.  For  ex- 
ample, if  one  submits  a  sweetened  must,  containing  nitrates 
or  some  ammoniacal  salts,  to  the  action  of  yeasts,  one  notices 
the  appearance  of  new  cells  and  consequently  the  formation 


2  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

of  protoplasmic  substances,  which  are,  from  a  chemical  point 
of  view,  very  complex  substances. 

When,  on  the  contrary,  one  subjects  albuminous  matters 
to  the  action  of  certain  ferments,  they  undergo  a  putrefactive 
fermentation,  and  it  is  seen  that  they  pass  through  a  series  of 
transformations. 

The  albuminous  matters  change  first  into  proteoses,  then 
into  peptones,  into  amides,  and  finally  into  ammonia,  hydro- 
gen sulphide,  oxalic  and  carbonic  acids. 

In  the  first  of  the  two  examples  which  we  have  just  given, 
we  apparently  see  a  work  exclusively  synthetic,  characterized 
by  the  formation  of  protoplasm  at  the  expense  of  sugars  and 
nitrates.  In  the  second  example  an  activity  absolutely  op- 
posed to  the  preceding  one  seems  to  be  exhibited. 

However,  the  phenomena  are  much  more  complex.  In 
the  first  example  the  formation  of  new  cells  of  yeast  is  ac- 
companied by  that  of  a  protein  matter,  the  protoplasm ;  but 
that  substance  does  not  persist  in  an  unchangeable  state :  on 
the  contrary,  it  is  constantly  destroyed,  hydrated,  and  trans- 
formed by  the  living  cells.  When,  therefore,  new  cells  are 
formed,  organized  matter  is  decomposed.  In  the  second  ex- 
ample, the  ferments  which  decompose  the  albuminoid  mat- 
ters and  transform  them  into  various  products  of  cleavage, 
multiply,  grow,  and  thus  carry  on  an  extensive  synthetical 
process  accompanying  the  process  of  decomposition. 

We  may  conclude  from  these  observations  that  the  living 
cell  works  constantly  in  two  different  ways,  analytically  and 
synthetically,  and  one  of  these  actions  is  more  apparent  than 
the  other  according  to  the  particular  case. 

Since  Wohler  accomplished  the  synthesis  of  urea,  it  has 
been  possible  to  produce  artificially  a  great  number  of  or- 
ganic substances.  Emil  Fischer  has  shown  us  the  course  to 
follow  for  carbohydrates,  and  it  has  been  possible  to  repro- 
duce artificially  almost  all  the  natural  sugars  which  are  found 
in  p1arts,  according  to  the  methods  which  he  has  indicated. 

While  we  do  not  actually  know  how  to  reproduce  albu- 


GENERAL   REMARKS.  3 

minoid  matters  synthetically,  the  works  of  Schiitzenberger 
have  at  least  made  known  the  manner  of  decomposition  of  all 
these  substances,  as  well  as  their  cleavage  products. 

Although  these  different  works  have  made  possible 
many  syntheses,  and  have  indicated  the  course  to  follow  to 
accomplish  others,  one  must  admit  that  there  is  a  great  dif- 
ference between  the  chemical  work  and  the  physiological 
work  of  cells. 

In  order  to  promote  chemical  reactions,  very  violent 
means  are  often  employed  in  the  laboratory.  Strongly 
acid  or  alkaline  solutions  are  used;  pressure  or  high  tem- 
peratures are  employed.  To  produce,  for  example,  the  phe- 
nomena of  oxidation,  use  is  made  of  reagents  such  as  nitric 
acid,  chromic  acid,  or  permanganates.  As  means  of  dehy- 
drating are  used  concentrated  sulphuric  acid,  anhydrous 
phosphoric  acid,  zinc  chloride,  etc.,  substances  which  destroy 
cells.  In  the  case  of  living  cells,  on  the  contrary,  the  re- 
actions occur  in  a  medium  either  neutral  or  weakly  acid  or 
alkaline;  the  temperature  is  always  very  moderate  and  al- 
most constant.  The  difference  between  these  two  condi- 
tions is  striking.  In  the  living  cell  bodies  react  which,  ac- 
cording to  our  ideas,  have  only  very  feeble  affinities;  one 
observes  at  the  same  time  that  substances  which  we  regard 
as  very  stable  are  readily  decomposed  in  the  interior  of  cells. 
The  affinity  of  chemical  bodies  appears  then  stronger  when 
they  are  in  the  presence  of  living  matter,  and  seems  to 
diminish  when  the  cells  are  destroyed. 

The  increase  of  potential  of  the  molecules  in  the  interior 
of  living  cells  is  generally  explained  by  the  intervention  of 
vital  energy.  The  reactions,  it  is  said,  are  produced  more 
easily,  owing  to  the  intervention  of  a  special  force,  the  vital 
energy,  which  increases  the  interior  energy  as  well  as  the 
aptitude  for  combinations  and  decompositions,  as  do  elec- 
tricity, magnetism,  light,  etc.  The  explanation  of  the  phe- 
nomena by  vital  energy  throws  little  enough  light  on  the 
subject.     In  brief,  it  reduces  itself  to  saying  that  reactions 


4  THE  ENZYMES  AND    THEIR   APPLICATIONS. 

are  favored  in  living  cells  by  physical  and  chemical  conditions 
peculiar  to  the  medium.  This  proposition  cannot  be  con- 
sidered as  an  explanation  of  intercellular  phenomena.  We 
cannot  really  understand  them  except  by  a  thorough  study  of 
the  media  in  which  they  are  produced.  A  careful  study  of 
these  conditions  shows  that  the  ready  occurrence  of  all  inter- 
cellular reactions  is  not  to  be  ascribed  to  a  single  cause ;  that 
the  affinity  is  sometimes  favored  by  a  purely  physical  con- 
dition and  sometimes  by  a  purely  chemical  one.  Certainly, 
we  are  far  from  knowing  all  the  conditions  which  favor  inter- 
cellular reactions,  but  it  has  been  possible  to  study  some 
among  them,  and  from  the  acquired  knowledge,  one  may 
conclude  that  whenever  one  observes  an  increase  of  cellular 
energy,  this  is  produced,  not  by  a  single  condition  common 
to  all  such  phenomena,  but  by  a  cause  strictly  determinable 
and  differing  in  different  cases. 

We  know  certain  reactions  in  which  chemical  affinity  in- 
creases because  of  purely  physical  conditions,  such  as 
osmosis,  which  continually  occurs  through  cell  membranes. 
In  other  cases  we  find  that  reactions  are  favored  in  the  cells 
by  the  presence  of  mineral  substances. 

The  decomposition  of  sodium  chloride,  for  example,  and 
the  formation  of  hydrochloric  acid  in  certain  cells  is  one  of 
those  phenomena  which  do  not  accord  at  all  with  the  general 
ideas  we  have  of  the  stability  of  certain  substances.  We 
know,  in  fact,  that  sodium  chloride  is  a  very  stable  substance, 
and  that  its  decomposition  in  the  cold,  in  a  medium  slightly 
acidified  by  weak  acids,  is  impossible.  Thus  the  de- 
composition of  sodium  chloride  in  the  cells  was  formerly* 
explained  by  the  intervention  of  vital  energy,  which  was  said 
to  render  the  body  less  stable  and  more  readily  decomposed. 
To-day  a  more  rational  explanation  is  given  to  the  phenom- 
enon: we  recognize  that  the  decomposition  of  sodium 
chloride  is  caused  simply  by  osmosis  and  independently  of 
vital  energy,  because  the  salt,  in  a  very  dilute  solution,  is 
dissociated.     In  the  cells  an  analogous  phenomenon  of  dis- 


GENERAL   REMARKS.  5 

sociation  must  be  produced,  and,  by  osmosis,  the  acid  must 
pass  through  the  cell  membrane  and  accumulate  in  a  certain 
•quantity.  Thus  the  acidity  is  seen  to  be  a  result  of  the  dis- 
sociation of  the  very  dilute  salt  solution  and  of  its  passage,  by 
osmosis,  through  the  cell  membrane.  This  is  a  very  striking 
example  of  a  physical  condition  favoring  reaction. 

The  most  convincing  example  of  the  intervention  of  min- 
eral substances  is  furnished  by  the  results  obtained  in  agricul- 
ture by  the  use  of  chemical  fertilizers.  When  one  places  at 
the  disposal  of  the  cells  relatively  small  quantities  of  phos- 
phate, the  quantity  of  protein  matter  produced  in  the  plants 
increases  considerably.  The  plant-cell  needs,  then,  to  carry 
on  its  synthetic  work,  the  presence  of  mineral  substances, 
which  form  organo-metallic  combinations ;  combinations 
which,  having  more  affinity  than  the  organic  substance  un- 
combined  with  the  mineral  substance,  enter  more  easily  into 
reaction. 

But  we  also  know  another  extensive  series  of  bio-chem- 
ical reactions  which  are  produced  without  intervention  of 
physical  factors  or  mineral  substances,  and  are  due  to  the 
prc~c"ce  of  chemical  substances  of  a  particular  nature  which 
we  call  enzymes.  The  study  of  these  substances,  of  their 
manner  of  secretion,  and  of  their  method  of  action  will  form 
the  object  of  the  present  work. 

The  enzymes,  soluble  ferments,  zymases,  or  diastases  are 
active  organic  substances,  secreted  by  cells,  and  have  the 
property,  under  certain  conditions,  of  facilitating  chemical 
reactions  between  certain  bodies,  without  entering  into  the 
composition  of  the  definite  products  which  result.  These 
substances  play  a  very  important  part  in  the  phenomena  of 
assimilation  and  of  disassimilation  of  foods.  In  fact,  most  of 
the  foods  which  occur  in  nature  at  the  disposition  of  men, 
lower  animals,  or  plants,  are  not  directly  assimilable;  they 
require  the  intervention  of  a  diastase  in  order  to  be  trans- 
formed into  substances  assimilable  and  suitable  for  the 
formation  of  new  tissues. 


6  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

Starch,  which  serves  in  the  nutrition  of  almost  all  living- 
creatures,  is  not  directly  assimilable,  and  in  the  higher  or- 
ganisms it  undergoes  various  transformations  before  it  can 
be  absorbed.  First  of  all,  it  encounters  the  enzymes  in  the 
saliva,  then  others  in  the  pancreatic  juice,  and  thus  it  is  trans- 
formed into  maltose  and  glucose,  foods  directly  suitable  for 
the  construction  of  tissues.  Meat,  milk,  and  white  of  egg 
must  also  be  transformed  under  the  influence  of  the  diastases 
before  becoming  assimilable.  These  substances  find  the 
enzymes  which  can  act  upon  them  in  the  gastric  and  pan- 
creatic juices. 

These  phenomena  which  are  observed  in  the  higher  or- 
ganisms are  also  met  with  in  the  vegetable  kingdom.  During- 
germination  and  florescence,  the  reserve  substances,  like 
starch,  cellulose,  fatty  substances,  and  proteid  matter,  are  in 
part  consumed  by  the  developing  plant.  But  this  utilization 
of  reserve  food  is  not  done  directly :  these  substances  must 
be  previously  transformed  by  the  diastases  into  assimilable 
products.  Let  us  examine,  for  example  the  phenomenon  of 
germination.  A  grain  of  barley  left  for  ten  or  fifteen  days-in 
darkness  loses  thirty  to  forty  per  cent  of  its  weight.  If  one 
determines  the  hydrogen  and  oxygen  in  the  grain  before  and 
after  germination,  one  finds  that  the  loss  of  these  two  ele- 
ments is  in  the  ratio  of  one  to  eight.  One  may  conclude 
from  this  that  oxygen  has  combined  with  hydrogen  to  form 
water.  On  the  other  hand,  if  one  determines  the  quantity  of 
carbonic  acid  formed,  it  is  found  that  it  corresponds  almost 
exactly  to  the  quantity  of  carbon  that  has  disappeared. 
There  would  be,  then,  combustion  of  carbon  and  formation 
of  carbonic  acid  on  one  hand,  formation  of  water  on  the  other 
hand,  and  the  phenomenon  would  appear  to  be  a  simple  oxi- 
dation. If  one  analyzes  the  reactions  more  closely,  it  is  seen 
that  germination  is  not  a  phenomenon  of  simple  oxidation, 
and  that  during  its  course  there  occurs  a  series  of  secondary 
reactions.  First  of  all,  there  appear  in  the  grain  diastases 
which  act  on  the  starch  and  the  cellulose  in  such  a  way  that 


GENERAL   REMARKS.  T 

little  by  little  these  two  substances  change  their  nature  as 
well  as  their  chemical  composition.  The  cellulose  is  dis- 
solved, the  starch  is  transformed  into  maltose,  part  of  which  is. 
oxidized,  and  part  changed  into  cane-sugar  by  the  tissue  of  the 
seed.  All  these  transformations,  as  well  as  the  oxidation  itself, 
are  produced  by  the  diastases  secreted  during  germination. 

One  can  follow  the  course  of  most  of  these  transforma- 
tions; for  example,  the  solution  and  the  transformation  of 
starch.  For  this  purpose  an  embryo  is  separated  from  the 
grain  and  made  to  develop  on  a  gelatinized  must  in  which 
starch  has  been  placed  in  suspension. 

By  observing  the  phenomenon  very  closely  and  by  ex- 
amining the  starch  under  the  microscope,  one  can  see  that 
the  grain  of  starch  loses  its  original  form,  that  it  is  corroded 
in  several  places,  that  it  then  liquefies  and  disappears.  In 
the  culture  liquid  one  finds  substances  which  did  not  exist  be- 
fore :  a  sugar,  and  a  nitrogenous  substance,  the  diastase, 
which  is  soluble,  capable  of  precipitation  by  alcohol,  and  can 
itself  produce  a  transformation  of  starch. 

In  the  assimilation  of  albuminoid  matter  by  cells,  there 

•  ... 

occurs  a  phenomenon  quite  analogous  to  the  assimilation  of 

carbohydrates.  The  albuminoid  substances  are  gradually 
transformed  by  the  active  substances  of  the  cells  into  pro- 
teids,  peptones,  and  finally  into  amides. 

We  have  said  above  that  the  diastases  play  an  extremely- 
important  part  in  the  phenomena  of  disassimilation.  The 
molecules  of  albuminoid  substances,  hydrated,  decomposed, 
and  transformed  by  the  enzymes,  are  regenerated  in  the  pres- 
ence of  the  protoplasm  of  the  cells,  by  a  process  of  dehydra- 
tion and  molecular  condensation.  The  reconstructed  mole- 
cules undergo  new  changes ;  they  are  again  hydrated,  decom- 
posed, and  at  the  same  time  gradually  oxidized.  In  this 
phase  of  the  transformation  the  albuminoid  molecule  is  de- 
composed into  urea,  glycogen,  fatty,  substances,  and  amides. 
These  transformations  are  also  in  great  part  due  to  the  active 
substances  secreted  by  the  cells. 


S  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

Finally,  the  enzymes  are  powerful  producers  of  heat ;  the 
reactions  caused  by  the  diastases  are  exothermic  reactions. 

Thus  a  molecule  of  urea  transformed  into  ammonium  car- 
bonate yields  8  calories.  A  molecule  of  glucose,  in  being 
transformed  into  carbonic  acid  and  alcohol,  sets  free  71 
calories.  Tripalmitin,  in  splitting  into  fatty  acid  and  glycerin, 
3rields  30  calories.  A  gram  of  albuminoid  matter  trans- 
formed into  urea  furnishes  4.6  Calories. 

It  is  seen  that  the  role  of  the  enzymes  as  producers  of 
heat  in  living  organisms  is  of  considerable  importance.  This 
heat,  set  free  by  exothermic  reactions,  is  then  utilized  by.  the 
cells  for  maintenance  as  well  as  for  the  construction,  of  their 
new  tissues. 

When  yeast  is  put  in  a  solution  of  saccharose,  there  is  first 
secreted  a  diastase  which  makes  the  medium  assimilable,  be- 
cause the  saccharose  cannot  be  directly  assimilated  by  the 
yeast.  There  is  produced  in  the  yeast  a  sucrase  which  trans- 
forms the  sugar  into  invert-sugar,  that  is  to  say,  into  dex- 
trose and  levulose.  The  cell  is  then  in  a  medium  favorable 
for  its  development:  it  can  utilize  the  nutritive  substances 
and  transform  them  into  tissues,  but  this  transformation  in- 
volves an  absorption  of  energy. 

On  the  one  hand  then,  the  yeast  has  need  of  energy  for 
the  maintenance  of  its  tissues ;  on  the  other,  the  heat  set  free 
by  the  transformation  of  cane-sugar  into  invert-sugar  is  not 
very  considerable  and  entirely  insufficient  to  produce  the  re- 
quisite energy.  The  yeast-cell,  therefore,  secretes  a  second 
diastase  acting  on  the  invert-sugar  much  more  powerfully, 
and  transforming  it  into  alcohol  and  carbon  dioxide.  These 
two  substances,  the  alcohol  and  the  carbon  dioxide, 
are  not  of  use  to  the  yeast ;  but  the  transformation  which  has 
produced  them  is  an  exothermic  reaction  which  furnishes  to 
the  cell  the  energy  which  it  needs  for  its  maintenance. 

An  example,  perhaps  still  more  striking,  is  the  trans- 
formation of  urea  into  carbonate  of  ammonium  by  special  fer- 
ments. 


GENERAL   REMARKS.  9 

If  these  ferments  are  cultivated  in  a  medium  containing 
urea  and  peptones,  it  is  found  that  the  cells  select  the  pep- 
tones as  tissue-building-  materials;  at  the  same  time  the  urea 
is  attacked  and  transformed  into  ammonium  carbonate. 
The  aim  of  this  second  transformation  is  to  furnish  the 
energy  necessary  to  the  cells  for  the  construction  and  the 
maintenance  of  their  tissues. 

YVe  observe  again  the  same  phenomenon  in  the  vegetable 
kingdom.  In  the  green  parts  of  plants,  under  the  influence 
of  the  sun's  rays,  the  carbonic  acid  is  constantly  decomposed ; 
formic  aldehyde  is  produced,  which  is  polymerized,  and  trans- 
formed into  different  carbohydrates.  Through  diffusion, 
these  carbohydrates  are  distributed  to  different  parts  of  the 
plants,  where  they  undergo  the  action  of  diastases  which  hy- 
drate them  and  decompose  them.  The  carbohydrates  are 
then  sources  of  heat,  which  is  set  free  by  their  decomposi- 
tion. The  products  of  decomposition  or  hydration  are  re- 
turned by  diffusion  to  the  green  parts,  where  they  can  again 
become  synthesized  and  consequently  store  up  heat. 

Thus  are  explained  the  migration,  hydrations,  and  dehy- 
drations of  the  carbohydrates  which  one  observes  in  these 
plants. 

We  may  conclude  from  all  these  facts  that  the  diastases 
are  substances  absolutely  indispensable  to  the  life  of  organ- 
isms, for  they  make  possible  the  construction  of  the  cellular 
tissues,  by  rendering  the  materials  assimilable  and  by  fur- 
nishing the  necessary  energy. 

BIBLIOGRAPHY. 

CI.  Bernard. — Lemons  sur  les  digestions.    Paris. 

V.  Kuhne. — Erfahrungen  und  Bemerkungen  uber  Enzyme  und  Fermented 

Physiologisches  Institut,  Heidelberg,  1878. 
Ad.  Mayer. — Die  Lehre  von  den  chemischen  Fermente,  1882. 
Duclaux. — Microbiologic     Dunod,  editeur.     Paris,  188,3,  p.  134. 
Armand  Gautier. — Leqons  de  chimie  biologique.     Paris,  1897. 
J.    Effront. — Actions   des   substances  minerales   et  des   diastases   sur  les 

cellules.     Moniteur  scientifique,   1894,  p.  562. 


CHAPTER  II. 

GENERAL    PROPERTIES. 

History  of  the  knowledge  of  enzymes. — Works  of  Reaumur  and  Spallan- 
zani, Kirchoff,  Dubrunfaut  and  Payen. — General  properties  of  dia- 
stases.— Means  of  distinguishing  a  diastatic  action  from  a  purely 
chemical  action. — Test  by  tincture  of  guaiacum.— :Law  of  proportion- 
ality in  diastatic  action. — Means  of  distinguishing  the  work  of  or- 
ganized ferments  from  diastatic  action. — Means  of  isolating  the  dia- 
stase from  the  medium  which  contains  it. — Chemical  composition  of 
enzymes. — Zymogenesis. — Method  of  action  of  diastases. 

The  first  ideas  concerning-  the  existence,  as  well  as  the 
action,  of  enzymes  belong  to  a  very  remote  period.  As 
early  as  the  beginning  of  the  sixteenth  century  the  phe- 
nomena of  digestion  claimed  much  attention  from  students. 
Opinion  on  this  subject  was  very  much  divided;  some  held 
that  digestion  was  a  purely  mechanical  work,  a  trituration  of 
substances  by  the  walls  of  the  stomach ;  others,  on  the  con- 
trary, explained  digestion  as  due  to  a  dissolving  and  trans- 
forming activity  of  the  juices  of  the  stomach. 

Reaumur  and  the  Abbe  Spallanzani  upheld  the  second  hy- 
pothesis and  performed  very  conclusive  experiments  to  con- 
firm their  theory.  Reaumur,  in  order  to  take  account  of  the 
influence  of  secretions  of  the  stomach,  caused  hawks  to 
swallow  little  metallic  tubes  pierced  with  holes  and  filled 
with  meat,  grain,  or  albumen.  He  examined  the  contents  of 
the  tubes  after  these  had  been  cast  out  and  found  that  the 
albuminoid  substances  alone  were  liquefied  and  transformed 
by  the  gastric  juice,  while  the  starchy  substances  had  not  un- 
dergone any  change. 

Abbe  Spallanzani  devoted  himself  to  the  study  of  the  gas- 


GENERAL   PROPERTIES.  ir 

trie  juice  and  its  action.  To  procure  active  secretions  he 
made  birds  of  prey  swallow  little  sponges  attached  to  strings. 
He  then  drew  back  the  sponges  saturated  with  gastric  juice. 
Spallanzani  first  succeeded  in  producing  artificial  digestion 
by  placing  meat  in  contact  with  the  liquid  squeezed  from  the 
sponges.      He  found  that  it  became  liquefied  and  changed. 

These  conclusive  experiments  which  throw  so  much  light 
on  the  phenomena  of  digestion,  as  well  as  on  the  role  of  the 
diastases,  unfortunately  were  not  appreciated  at  their  true 
value.  They  did  not  succeed  in  convincing  the  scientific 
world,  and  at  the  beginning  of  the  nineteenth  century  the 
phenomena  of  digestion  were  still  interpreted  in  different 
ways.  Certain  scholars  maintained  that  the  gastric  juice 
"had  no  ,such  constant  character,  and  that  the  nature  and 
properties  of  the  secretion  depended  especially  on  the  foods 
absorbed.  These  differences  in  the  interpretation  of  the 
phenomena  of  digestion  retarded  the  study  of  enzymes,  al- 
though it  was  already  greatly  advanced  by  the  works  of 
Reaumur  and  Spallanzani.  It  was  not  until  nearly  two  cen- 
turies after  their  publication  that  the  question  of  active  sub- 
stances secreted  by  the  cells  again  became  prominent. 

It  is  very  curious  to  find  that  it  is  the  study  of  brewing 
which  has  led  to  the  greatest  discoveries  of  this  century.  It 
was  by  the  study  of  the  beer-yeasts  that  Pasteur  established 
definite  arguments  against  the  theory  of  spontaneous  genera- 
tion. It  is  also  by  the  study  of  the  raw  materials  of  the 
brewery,  notably  malt,  that  Dubrunfaut  laid  the  foundation 
for  the  study  of  enzymes. 

The  work  of  Dubrunfaut  is  connected  with  an  observa- 
tion made  by  Kirchoff  in  1814.  This  distinguished  scholar, 
who  was  the  first  to  study  the  transformation  of  starch  by 
acids,  had  noticed  that  fresh  gluten  can  act  under  certain 
conditions  on  starch,  dissolve  it,  and  transform  it  into  a  sac- 
charine substance.  This  experiment  was  taken  up  again  by 
Dubrunfaut,  who,  in  a  long  and  masterly  study,  demon- 
strated that  the  activity  of  gluten  is  due  to  the  presence  of  a 


12  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

small  quantity  of  active  substance  originating  in  the  raw 
grain.  He  demonstrated  that  this  diastase  is  soluble  in 
water,  that  the  ungerminated  grain  contains  very  little  of  itr. 
and  that  germination  develops  it.  He  explained  the  mode 
of  action  of  this  substance  and  the  conditions  under  which  the 
maximum  effect  is  produced ;  he  proved  finally  that  the 
sugar  prepared  from  starch  by  the  aid  of  this  substance  is  not 
identical  with  the  glucose  which  Kirchoff  obtained  by  the 
action  of  the  acid.  It  is  in  the  works  of  Dubrunfaut,  pub- 
lished in  1822,  that  we  find  for  the  first  time  a  scientific  study 
of  the  diastases  as  well  as  precise  data  concerning  their  mode 
of  action. 

Payen  took  up  again  the  work  of  Dubrunfaut,  from  whom, 
he  unjustly  withheld  the  credit  for  the  discovery  of  the  dia- 
stases of  malt.  In  studying  the  properties  of  an  infusion  of 
malt,  Payen  recognized  that  the  active  substance  can  be  pre- 
cipitated from  its  solution  by  alcohol,  and  the  precipitate 
thus  obtained  exhibits  all  the  properties  which  the  liquid 
itself  possessed.  This  experiment  has  played  a  considerable 
part  in  the  discovery  of  enzymes,  because  Payen  had  thus 
found,  at  once,  a  general  property  of  diastases  and  a  general 
means  of  isolating  them. 

By  this  method  have  been  isolated  the  active  substances 
of  the  gastric  juice  and  of  the  pancreatic  juice,  as  well  as  the 
enzymes  acting  on  fatty  substances  and  glucosides. 

GENERAL  PROPERTIES  OF  ENZYMES. 

We  have  just  seen  that  the  enzymes  are  precipitated  from, 
their  solutions  by  alcohol,  but  it  is  necessary  to  add  that, 
while  all  the  enzymes  are  precipitated  by  very  concentrated 
alcohol,  they  are  more  or  less  soluble  in  dilute  alcohol. 
Since  the  discoveries  of  Payen,  there  have  been  recognized  a 
certain  number  of  other  properties  more  or  less  characteris- 
tic of  enzymes.  Enzymes  are  soluble  in  water,  are  thrown 
down  from  their  solutions  by  indifferent  precipitates,  become 


GENERAL   PROPERTIES.  IS 

fixed  on  different  substances  as  silk  and  fibrin,  and  are 
somewhat  resistant  to  poisonous  substances.  Enzymes  lose 
their  activity  at  a  temperature  in  the  neighborhood  of  ioo°. 
The  greater  part  of  them  decompose  hydrogen  peroxide. 
They  are  also  characterized  by  the  fact  that,  under  given 
conditions,  the  action  which  they  produce  is  proportional 
to  their  quantity.  All  these  properties  are,  however,  far 
from  being  distinctive ;  many  other  substances  than  diastases, 
possess  one  or  another  of  them.  The  most  characteristic 
property  of  an  enzyme  is  its  special  mode  of  operation. 

Let  us  consider  each  of  the  properties  which  we  have  just 
cited.  We  have  seen,  first  of  all,  that  enzymes  are  precipi- 
tated by  alcohol ;  as  they  are  more  or  less  soluble  in  dilute 
alcohol,  the  quantity  of  alcohol  which  is  necessary  to  precipi- 
tate the  enzymes  from  an  aqueous  solution  will  not  always  be 
the  same.  For  certain  diastases,  the  fibrin  ferment  for  ex- 
ample, it  will  be  sufficient  to  put  in  the  solution  10  to  15  per 
cent  of  alcohol,  which  constitutes  a  minimum  proportion. 
For  others,  as  the  coagulative  ferment  of  milk,  it  is  necessary 
to  add  a  large  proportion  of  alcohol,  in  order  to  have  a  liquid 
containing  80  to  90  per  cent. 

But  if  all  the  enzymes  are  precipitated  by  alcohol,  they 
are  likewise  all  destroyed  by  this  same  agent.  By  a  pro- 
longed contact  of  the  diastase  with  the  alcohol,  the  active 
substance  is  transformed,  becomes  insoluble  and  inactive. 
So,  if  one  precipitates  an  enzyme  by  concentrated  alcohol, 
the  action   must  be  stopped  as  quickly  as  possible. 

Enzymes,  from  the  point  of  view  of  solubility  in  water, 
present  noteworthy  differences.  We  know  of  diastases  which 
dissolve  very  easily  and  of  others,  on  the  contrary,  which  re- 
quire for  their  solution  a  large  quantity  of  water.  Moreover, 
remembering  that  active  substances  are  fixed  with  ease  on 
different  bodies,  it  is  easy  to  understand  that  the  same  sub- 
stance can  be  presented  in  soluble  or  insoluble  form. 

The  precipitation  of  enzymes  by  "  dragging  down  " 
(mechanical  precipitation)  can  be  easily  effected.     One  adds 


14  THE  ENZYMES  AND   THEIR.  APPLICATIONS. 

to  a  filtered  and  clear  infusion  of  malt  a  very  dilute  solution 
of  sodium  phosphate,  then  a  solution  of  a  calcium  salt;  there 
is  produced  in  the  liquid  a  precipitate  of  calcium  phosphate 
which  finally  settles  at  the  bottom  of  the  vessel ;  the  clear 
liquid  is  decanted,  the  precipitate  placed  on  a  filter,  washed 
with  a  little  water,  and  a.  powder  is  obtained  which  possesses 
all  the  properties  of  the  infusion  of  malt.  This  powder,  for 
instance,  acts  on  starch  and  produces  maltose,  as  does  the 
malt  from  which  it  has  been  extracted.  This  method  makes 
it  possible  to  obtain  all  the  enzymes  contained  in  an  infusion. 
Only  one  condition  is  necessary, — that  the  substances  em- 
ployed for  the  precipitation  shall  be  harmless  to  the  diastase. 
Good  results  are  obtained,  for  instance,  in  making  use  of 
magnesium  carbonate  or  aluminium  hydrate. 

We  have  seen  that  the  diastases  become  fixed  on  different 
substances.  Thus  a  piece  of  fibrin  placed  in  a  solution  of 
gastric  juice  becomes  impregnated  with  the  active  substance 
in  such  a  way  that  the  diastase  cannot  any  longer  be  removed 
by  washing. 

If,  after  having  withdrawn  this  fibrin  from  the  infusion 
and  washed  it  to  remove  as  far  as  possible  every  trace  of  ac- 
tive substance,  it  is  placed  in  water  at  a  suitable  temperature, 
the  piece  of  fibrin  dissolves.  It  is  evident  that  this  transfor- 
mation of  the  protein  matter  is  due  to  the  fact  that  the  active 
substance  is  fixed  on  the  fibrin  like  a  dye.  It  is  not  more- 
over necessary  for  the  diastase  to  act  on  a  substance  to 
become  fixed  on  it.  For  example,  if  one  places  some  pieces 
of  silk  in  gastric  juice,  they  are  impregnated  with  active  sub- 
stance, although  the  diastase  does  not  act  at  all  on  the  silk. 

Most  diastases  are  insensible  to  the  action  of  certain  sub- 
'stances,  such  as  hydrocyanic  acid  and  chloroform,  which 
paralyze  the  vital  activity  of  cells.  If,  for  example,  yeast  is 
put  in  a  solution  of  cane-sugar  in  the  presence  of  chloroform, 
the  yeast  remains  quiescent  and  does  not  reproduce ;  how- 
ever, the  cane-sugar  is  still  transformed  into  invert-sugar. 
The  diastase  continues,  then,  to  be  secreted  by  the  cells  and 


GENERAL   PROPERTIES.  15 

to  do  chemical  work,  while  the  cellular  activity,  properly 
speaking,  is  paralyzed  by  the"  chloroform. 

From' these  experiments  it  appears  that  enzymes  are  not 
sensitive,  either  to  the  action  of  antiseptics,  or  to  that  of  sub- 
stances which  are  opposed  to  the  vital  action.  But  the  rule 
is  not  universal.  We  know,  in  fact,  of  several  enzymes  which 
are  extremely  sensitive  to  chloroform,  ether,  and  thymol,  as 
well  as  to  hydrocyanic  acid. 

In  reality,  the  various  diastases  differ  considerably  among 
themselves  as  to  their,  nature,  and  as  to  their  sensitiveness 
towards  the  different  reagents. 

The  diastases  of  malt  as  well  as  the  active  substances  of 
the  yeasts  transforming  the  cane-sugar  into  invert-sugar,  are 
very  resistant  enzymes,  and  much  less  sensitive  than  the  cells 
which  elaborate  them. 

These  same  diastases  sometimes  occur  in  other  more  re- 
sistant living  cells,  because  there  exists  among  cells,  as 
among  these  diastases,  considerable  differences  in  sensitive- 
ness to  reagents.  There  are,  then,  cases  where  antiseptics 
attack  enzymes  before  acting  on  the  cells,  and  other  cases 
where  the  reverse  is  the  case.  The  yeast  which  we  have  just 
cited  furnishes  an  example  of  the  relative  sensitiveness 
of  diastases  to  antiseptics.  ,  As  a  matter  of  fact  it  is  known 
that  beer-yeast  contains,  in  addition  to  the  diastase 
changing  cane-sugar  into  invert-sugar,  a  second  enzyme 
which  transforms  the  invert-sugar  into  alcohol.  The  absence 
of  fermentation  in  presence  of  chloroform  proves  that,  of  the 
two  diastases  contained  in  the  yeast,  one  is  destroyed  by  the 
antiseptic  while  the  other  resists  it. 

The  greater  or  less  sensitiveness  of  diastases  to  the  action 
of  antiseptics,  and  to  those  substances  paralyzing  the  vital 
activity,  can  be  utilized  to  exclude  the  activity  of  micro-or- 
ganisms during  diastatic  action. 

When  one  studies,  for  example,  the  saccharification  of 
starch  or  the  transformation  of  meats  by  enzymes,  one  may 
often  be  led  into  error  by  the  invasion  of  organisms  which 


1 6  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

produce  the  same  effect  as  the  diastase  whose  action  is  being 
studied.  In  this  case,  a  small  amount  of  antiseptic,  for  ex- 
ample, a  few  drops  of  chloroform,  is  put  in  the  liquid  which  is 
kept  under  observation,  and  interference  from  ferments  is 
prevented.  Only,  in  the  case  of  certain  enzymes  more  sen- 
sitive than  others,  it  is  necessary  to  use  other  means  for  pre- 
venting the  action  of  organized  ferments,  because  the  en- 
zyme itself  would  be  destroyed  by  the  antiseptic.  This  pre- 
caution is  especially  necessary  when  one  is  studying  the  ac- 
tion of  enzymes  yet  unknown;  in  this  case  a  negative  result 
may  be  due  to  the  presence  of  an  antiseptic. 

The  action  of  heat  on  the  enzymes  is  an  ex- 
tremely important  point  and  one  which,  better  than 
any  other,  may  serve  to  characterize  a  diastatic  ac- 
tion. In  general,  with  a  certain  number  of  exceptions, 
enzymes  exert  their  action  slowly  at  a  temperature  of  o° ; 
often  at  this  temperature  the  effect  they  produce  is  impercep- 
tible. If  one  gradually  increases  the  temperature  to  400  the 
reaction  is  intensified;  from  400  to  500  there  is  a  marked 
increase  in  intensity, — it  is  generally  at  this  temperature  that 
the  diastase  attains  its  maximum  activity, — above  50°  the 
activity  diminishes ;  at  8o°  a  considerable  weakening  is  pro- 
duced, and  finally,  above  90°  the  diastase  is  wholly  destroyed. 
The  different  diastases  are  characterized  by  their  optimum 
temperatures,  that  is,  by  the  temperature  at  which  they  give 
their  maximum  of  action.  This  temperature  varies  quite 
considerably  in  different  enzymes,  and  this  variation  consti- 
tutes a  property  which  admits  of  differentiation. 

But  the  property  of  enzymes  which  is  most  useful  in 
studying  them,  is  the  facility  with  which  they  are  destroyed 
from  900  to  ioo°  in  the  presence  of  water.  Some  diastases, 
when  they  are  in  a  completely  dry  state,  can  stand  a  tempera- 
ture of  900  and  even  more ;  but  all  enzymes,  without  excep- 
tion, lose  their  activity  when  their  aqueous  solution  is  brought 
to  the  neighborhood  of  ioo°.  This  property  is  utilized  for 
distinguishing  diastatic  from  purely  chemical  action. 


GENERAL   PROPERTIES.  17 

When  one  puts  an  infusion  of  yeast  in  a  solution  of  sac- 
charose, the  transformation  of  saccharose  into  invert-sugar 
takes  piace.  But  it  is  not  to  be  inferred  that  this  is  neces- 
sarily diastatic  action,  for  the  transformation  may  be  due, 
either  to  the  acidity  of  the  must,  or  to  some  other  chemical 
agent. 

To  prove  that  there  exists  in  a  yeast  an  active  substance, 
a  double  experiment  is  necessary.  One  must  treat  equal 
quantities  of  sugar  equally  diluted,  during  the  same  length 
of  time  and  at  the  same  temperature,  on  the  one  hand  with 
a  certain  quantity  of  infusion  of  yeast,  and  on  the  other  hand 
with  an  equal  quantity  of  the  same  infusion,  which  has  been 
previously  heated  to  ioo°  for  several  minutes,  and  then 
cooled  again. 

If  the  same  result  is  obtained  in  the  two  experiments,  one 
may  conclude  that  the  transformation  is  not  clue  to  an  active 
substance  contained  in  the  infusion  under  examination.  On 
the  contrary,  the  activity  of  a  diastase  becomes  evident,  if  in 
the  experiment  with  the  heated  infusion  inversion  is  not  ob- 
tained, while  from  the  action  of  the  infusion  not  heated  a 
transformation  is  observed. 

The  fact  that  diastases  are  destroyed  at  ioo°  relates  them 
in  a  striking  manner  to  living  organic  matter. 

We  have  said  above  that  when  an  enzyme  is  put  in  solu- 
tion in  the  presence  of  hydrogen  peroxide,  the  latter  is  de- 
composed. To  demonstrate  this  reaction,  one  makes  use  of 
an  alcoholic  solution  of  guaiacum.  Generally  2  or  3  cubic 
centimetres  of  tincture  of  guaiacum  are  taken  ;  a  few  drops  of 
hydrogen  peroxide  are  added  and  then,  drop  by  drop,  the 
liquid  in  which  an  enzyme  is  supposed  to  exist.  In  the  pres- 
ence of  an  enzyme  the  red  liquid  becomes  a  very  intense  blue. 

This  coloring  is  due  to  the  transformation  of  the  guaia- 
conic  acids  into  guaiacosonide,  a  dye.  The  reaction  with 
tincture  of  guaiacum  is  extremely  sensitive  :  one  can  produce 
it  with  exceedingly  small  quantities  of  active  substance. 

It  must  not  be  forgotten  that  tincture  of  guaiacum  loses 


1 8  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

in  time  the  property  of  giving  color,  and  that  the  best  way  is. 
to  make  the  tincture  before  the  experiment  by  grinding 
powder  of  guaiacum  with  alcohol ;  moreover,  the  use  of  this 
reagent  always  offers  certain  difficulties :  it  must  be  noted 
that  the  coloring  matter  formed  is  not  very  stable,  that  it  is 
decomposed  by  heat  as  well  as  by  different  chemical  re- 
agents. A  slight  alkalinity  or  even  a  slight  acidity  is  suf- 
ficient to  prevent  the  production  of  coloration,  and  it  fol- 
lows that  it  is  necessary  to  take  some  precautions  when  using 
this  reagent.  It  is  well  first  to  neutralize  exactly  the  hydro- 
gen peroxide  used,  for  this  is  generally  very  acid;  it  is  also 
well  to  measure  the  degree  of  acidity  or  alkalinity  of  the 
liquid  containing  the  diastase  to  be  studied,  then  to  neu- 
tralize it  when  it  is  clearly  alkaline  or  clearly  acid. 

The  coloration  produced  by  guaiaconic  acid  is  not 
destroyed  by  acetic  acid  and  it  is  often  of  advantage,  when 
working  with  an  absolutely  neutral  solution,  to  acidify  it  with. 
a  drop  of  dilute  acetic  acid. 

The  reaction  of  the  tincture  of  guaiacum  is  of  much  use 
in  the  investigation  of  enzymes.  However,  this  reagent  is 
not  absolutely  reliable,  for  the  coloration  observed  in  a  solu- 
tion may  be  due  to  other  bodies  than  enzymes.  Moreover, 
if  a  reaction  is  not  obtained,  one  must  not  conclude  that  the 
liquid  does  not  contain  an  active  substance,  for  the  coloration 
may  be  prevented  by  different  substances  which  may  be  pres- 
ent with  the  enzymes  in  the  liquid  tested. 

Moreover,  enzymes  are  known  which  do  not  give  colora- 
tion with  guaiacum  and,  on  the  other  hand,  diastases  which, 
after  having  been  submitted  to  certain  influences,  lose  this 
property  without  always  losing  their  activity.  Thus,  at  a 
high  temperature,  certain  enzymes  no  longer  give  coloration 
with  guaiacum,  although  the  active  substance  is  not 
destroyed.  With  other  diastases  the  property  of  coloring 
tincture  of  guaiacum  disappears  in  a  prolonged  contact  with 
hydrogen  peroxide,  contact  which  has  no  influence  on  the 
activity  of  the  diastase.     However,  no  enzymes  are  known 


GENERAL   PROPERTIES.  19 

which,  after  having  lost  their  activity  by  the  action  of  chem- 
ical or  physical  agents,  still  give  a  coloration  with  tincture 
of  guaiacum. 

It  follows  that,  to  use  the  reaction  of  guaiacum,  one  must, 
as  with  experiments  on  the  effect  of  heat,  make  double  ex- 
periments with  the  fresh  infusion  on  the  one  hand,  and  with 
the  infusion  heated  to  100°  on  the  other  hand.  When  the 
fresh  infusion  produces  a  coloration  and  the  infusion  after 
being  heated  does  not,  one  may  be  confident  of  the  presence 
of  a  diastase. 

Further,  tincture  of  guaiacum  gives  a  blue  coloration, 
with  a  whole  group  of  diastases,  without  hydrogen  peroxide. 
In  this  case,  the  guaiacum  justifies  not  only  the  inference  that 
a  diastase  is  present  in  a  solution,  but  gives  further  informa- 
tion ;  the  reaction  of  guaiacum  without  hydrogen  peroxide 
being  possible  only  with  an  oxidizing  enzyme. 

Tincture  of  guaiacum  can  also  be  of  great  service  when 
plant  enzymes  are  being  studied. 

It  often  happens  that  diastases  contained  in  vegetable 
cells  are  changed  or  destroyed  as  the  result  of  maceration  in 
water,  on  account  of  the  dissolving  out,  from  the  cells,  of 
extractive  substances  which  destroy  the  enzymes.  In  this 
case  one  must  look  for  the  diastases,  not  in  the  solution,  but 
in  the  cells  themselves. 

For  this,  very  small  sections  are  made  which  are  intro- 
duced either  in  the  pure  tincture  of  guaiacum  or  in  a  solution 
of  guaiacum  added  to  hydrogen  peroxide.  The  cells  contain- 
ing the  active  substance  are  colored  blue. 

It  is  often  very  difficult  to  distinguish  a  diastatic  from  a 
strictly  cellular  action.  If  it  is  noticed  that  a  certain  liquid  is 
capable  of  producing  chemical  changes  in  certain  substances, 
one  is  led  to  think  that  an  enzyme  is  present  if  the  same  liquid 
after  boiling  has  not  the  same  power.  But  in  reality  there  is 
nothing  to  prove  in  this  case  that  the  action  observed  is  really 
a  strict  diastatic  phenomenon,  for  certain  organized  ferments 
may  have  caused  it. 


20  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

To  determine  exactly  whether  organized  or  soluble  fer- 
ments are  present,  recourse  may  be  had  in  certain  cases  to  a 
nitration  by  means  of  a  porous  filter  which  is  capable  of  re- 
taining the  organisms.  If  the  filtered  liquid  is  still  active, 
one  may  conclude  that  the  transformations  noticed  are  really 
diastatic  phenomena.  But  the  contrary  does  not  prove  the 
absence  of  an  enzyme  in  the  solution,  for  all  enzymes  are 
more  or  less  retained  by  the  porous  substance  of  the  filter, 
and  certain  among  them  do  not  pass  through  at  all. 

It  is  in  the  proportionality  which  exists  between  the  quan- 
tity of  diastases  employed  and  the  quantity  of  substance 
transformed  by  these  diastases  that  a  certain  proof  is  found 
of  the  existence  of  a  diastase.  The  law  of  proportionality  is 
not,  however,  an  absolute  law.  With  an  infinitely  small 
quantity  of  enzyme  one  can  transform  a  very  considerable 
quantity  of  substance  on  condition  that  the  action  is  allowed 
to  be  continued  for  a  long  time  under  such  conditions  that 
the  enzyme  is  not  destroyed  by  the  physical  and  chemical 
agents  of  the  medium.  However,  at  the  beginning  of  the 
action,  especially  if  one  employs  a  very  small  quantity  of 
active  substance  and  a  large  quantity  of  passive  substance, 
one  notes  a  fixed  ratio  between  the  quantities  of  enzyme  em- 
ployed and  of  substance  transformed. 

It  is  under  these  conditions  only  that  the  law  of  propor- 
tionality -,can  be  verified.  If  one  adds,  for  example,  to  ioo 
cubic  centimetres  of  a  ten  per  cent  sugar  solution  a  slight 
quantity  of  sucrase,  for  example  a  cubic  centimetre  of  an  in- 
fusion of  yeast,  and  if  the  act-ion  is  stopped  after  one  hour,  it 
is  found  that  a  part  of  the  sugar  has  been  transformed.  If  in 
a  similar  liquid  under  the  same  conditions  of  dilution  and 
temperature,  \  cubic  centimetre  of  the  same  solution  of  su- 
crase be  added,  one  finds  that  the  quantity  of  sugar  inverted 
is  very  nearly  half  of  the  quantity  transformed  in  the  preced- 
ing experiment. 

If  instead  of  diastases  one  employs  organized  ferments 
capable  of  effecting  the  same  transformation,  one  never  ob- 


GENERAL   PROPERTIES.  21 

serves  a  proportionality  between  the  quantity  employed  and 
the  result  obtained.  A  double  quantity  of  organized  fer- 
ments does  not  transform  twice  as  much  sugar.  There  is 
evidently  in"  the.  second  case  a  larger  quantity  of  sugar  in- 
verted, but  this  quantity  is  not  double.  The  proportionality 
between  thequantities  of  diastase  employed  and  of  substance 
transformed  is  of  great  use,  especially  when  one  suspects  the 
presence  of  organized  ferments  in  an  active,  liquid. 

Chemical  Composition  of  Enzymes. — Now  that  we  know 
the  means  of  recognizing  the  presence  of  diastases  in  a  liquid, 
let  us  study  closely  the  chemical  composition  of  enzymes. 

The  elementary  analysis  of  enzymes  gives  discordant 
figures  for  the  different  known  kinds,  and  sometimes  even  for 
the  same  diastase  different  authors  have  found  very  different 
results.  This  fact  may  arise  because  the  materials  submitted 
to  analysis  are  never  pure,  but  mixtures  of  different  sub- 
stances. It  may  be  also  that  the  enzymes  really  differ  in 
their  composition,  and  this  should  not  surprise  us  since  they 
are  bodies  which  act  in  various  ways  and  upon  very  different 
substances.  The  composition  of  some  enzymes  is  here 
given: 


Carbon. 

Hydrogen 

Nitrogen. 

Sulphur. 

Ash. 

Experimenters. 

Diastase    from 

Pancreatin  .... 

White    of    egg 
uncoagulated 

T45-68 

J  47-57 
1  46.66 

6.9 
6.49 

7-35 

4-57 

5-14 

10.41 

16.53 
11.86 

4-3° 
6. 

9-3° 
11.52 
14.20 
13.81 

16.55 
17.8 

inoid  Subs 
15.8 

O 

6.08 
3.16 
4-79 

Krauch. 

Zulkowski, 

Lintner. 

Wroblewski. 

Hiifner. 

Mayer. 

Brauth. 

Donath. 

Brucklau. 

Schmid. 

Hiifner. 

Loenid. 

Schmid. 

Dumas. 

43-1 
( 

7-8 

6.1 

•  43.90 

I  40-50 

(43.06 

j  48 . 80 

43.6 

52.75 

53-2 

53-7 

8.4 
6.9 
7.2 
7-i 
6-5 
7-5 
6.7 

Album 
7-i 

O.63 
1-25 

1-3 

0.88 

7.04 
17-7 

ances. 
1.8 

22  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

By  examining  the  percentage  of  nitrogen  of  the  several 
enzymes  whose  composition  we  give  in  the  table  above,  we 
notice  that  certain  diastases,  as  pepsin,  contain  great  quanti- 
ties, and  approach  albuminoid  substances  in  composition. 
We  see,  on  the  contrary,  that  other  enzymes  as  invertin  have 
a  much  smaller  nitrogen  content.  In  the  group  of  the  oxi- 
dases there  are  also  enzymes,  quite  recently  discovered, 
which  appear  to  be  absolutely  lacking  in  nitrogen.  These 
latter  substances  are  analogous  rather  to  the  gums. 

We  have  just  said  that  the  non-agreement  of  results  may 
be  due  to  impurity  of  substances  analyzed.  In  reality,  the 
methods  which  are  followed  for  separating  the  enzymes  from 
the  media  which  contain  them  cannot  furnish  pure  sub- 
stances. 

Usually  the  diastase  is  obtained  from  the  cells  by  ex- 
tracting with  water  and  then  precipitating  with  alcohol  the 
infusion  obtained.  In  liquids  which  have  been  in  the  pres- 
ence of  protoplasmic  substances,  there  is  always  a  large 
quantity  «of  matter  which  can  be  precipitated  by  alcohol,  and 
the  results  obtained  are  of  necessity  mixtures  of  different 
bodies.  When  it  is  proposed  to  purify  the  precipitates  by 
dissolving  them  and  reprecipitating,  one  obtains  many 
bodies  of  a  stable  composition,  but  almost  entirely  destitute 
of  all  active  power. 

In  enzymes  one  always  finds  a  great  quantity -of  inorganic 
salts,  particularly  calcium  phosphate,  in  very  varied  propor- 
tions. If  the  method  of  mechanical  precipitation  is  em- 
ployed to  isolate  the  diastase,  the  result  is  the  same:  after 
the  precipitation  bodies  containing  many  impurities  are 
found.  Furthermore,  in  precipitating  a  diastase  in  an  active 
liquid,  one  always  runs  the  risk  of  obtaining  a  mixture  of  dif- 
ferent diastases  and  not  one  alone.  It  then  becomes  abso- 
lutely impossible  to  separate  them  from  each  other,  because 
their  insolubility  in  alcohol  is  not  such  that  they  can  be 
separated  by  precipitation.  Thus,  when  barley-malt  is 
steeped  in  water,  there  is  obtained  in  the  liquid  a  whole  series 


GENERAL   PROPERTIES.  25 

of  active  substances  which  are  precipitated  together  by- 
alcohol  or  by  other  substances  which  can  drag  them  down. 

The  diastases  which,  according  to  the  analyses,  most 
nearly  approach  proteids  in  composition,  still  differ  consider- 
ably from  these  substances. 

Enzymes  do  not  give  all  the  color  reactions  of  proteids. 
Bodies  of  this  class  cannot  diffuse  through  a  parchment  mem- 
brane, while  diastases  are  capable  of  doing  it,  although  with 
some  difficulty.  Diastases  act  differently  from  proteids. 
These  latter  bodies  can  be  assimilated  by  cells,  while  the  dia- 
stases cannot  be.  The  salivary  and  pancreatic  enzymes 
never  serve  as  reserve  substances.  Though  stored  in  the 
cells  during  the  period  of  normal  nutrition,  these  substances 
are  rejected  in  time  of  starvation.  According  to  Beijerinck, 
amylase  cannot  replace  in  a  nutritive  medium  either  carbohy- 
drates or  nitrogenous  substances,  the  yeasts  and  the  bacteria 
absolutely  refusing  to  be  nourished  by  it. 

Zymogenesis. — Enzymes  are  produced  by  certain  special 
cells.  According  to  Hufner  they  are  formed  by  the  oxida- 
tion of  albuminoid  substances.  This  theory  is  attacked  by 
Wroblewsky,  who  considers  diastases  as  proteoses. 

There  are  as  yet  very  few  data  on  the  manner  of  forma- 
tion of  enzymes.  In  most  cases  one  can  only  observe  their 
presence  when  they  have  acquired  all  their  properties;  in 
some  isolated  instances  the  presence  of  a  non-active  sub- 
stance capable  of  becoming  a  ferment  by  suitable  treatment 
has  been  observed. 

Thus  the  gastric  mucous  membrane,  when  soaked  in 
water,  yields  a  liquid  which  does  not  coagulate  milk ;  but  this 
liquid  acquires  that  property  when  one  adds  1  per  cent  of 
hydrochloric  acid,  and  preserves  its  activity  even  after 
neutralization.  Fresh  pancreatic  tissue  yields  in  water  a 
substance  acting  very  slowly  in  the  presence  of  a  slight 
quantity  of  acid.  The  activity  of  this  liquid  can  be  acceler- 
ated by  passing  through  it  a  current  of  oxygen,  or  by  intro- 
ducing into  it  hydrogen  peroxide. 


24  THE  ENZYMES  AND    THEIR   APPLICATIONS. 

These  substances,  capable  of  becoming  active,  are  called 
zymogens  (proferments,  proenzymases),  and  the  transforma- 
tion of  the  zymogen  into  the  ferment  is  called  according  to 
Arthus.:  zymogenesis.  It  is  very  probable  that  most  en- 
zymes come  from  zymogens,  and  that  the  phenomena  of 
zymogenesis  are  as  frequent  as  the  phenomena  of  destruction 
of  the*  diastase,  called  zymolysis. 

Manner  of  Action  of  Diastases. — The  chemical  analysis 
of  an  enzyme  is  not  sufficient  to  characterize  it.  To  deter- 
mine exactly  the  characteristics  belonging  to  a  diastase,  one 
must  observe  its  manner  of  action,  the  chemical  change  it  can 
produce,  and  especially  the  substances  on  which  it  can  act. 

Diastases  can  induce,  according  to  their  nature,  very  dif- 
ferent chemical  reactions.  Some  have  a  hydrating  action, 
that  is  to  say,  they  can  cause  one  or  more  molecules  of  water 
to  unite  with  the  substances  on  which  they  act.  We  can  cite 
for  example  the  transformation  of  saccharose  into  dextrose 
and  levulose. 

C12H22O10  +  H20  =  C6H1206  +  C6H1206. 

Saccharose.  Water.  Dextrose.  Levulose. 

Another  series  of  enzymes  acts  on  the  contrary  like  oxidizing 
agents.  An  example  of  this  class  is  the  transformation  of 
hydroquinone  into  quinone. 

C«H*<OH  +  O  =  H2°  +  C6H4<?« 

Finally,  other  enzymes  act  only  on  the  molecules  by  decom- 
posing them  without  producing  hydration  or  oxidation,  only 
causing  a  molecular  change  in  the  substance.  It  is  thus  that 
the  diastase  of  yeast  which  produces  alcoholic  fermentation 
gives  rise  to  a  simple  molecular  decomposition  without  hy- 
dration. 

C6H1206  =  2C02  +  2C2H5OH. 

Glucose.  Sggjfc  Alcohol. 

Thus,  in  the  example  which  we  have  cited  of  the  trans- 
formation of  saccharose  into  glucose  and  levulose,  the  mole- 


GENERAL   PROPERTIES.  25 

cnle  of  cane-sugar  is  found  to  be  hydrated  and  decom- 
posed. 

This  decomposition  of  the  molecules  with  hydration  also 
takes  place  in  the  transformation  of  glucosides  by  diastases. 
The  complex  molecule  of  the  glucosides,  becoming  hydrated, 
divides  into  two  parts,  yielding  glucose  and  the  body  with 
which  it  was  combined. 

One  observes  the  same  phenomenon  in  the  action  of  dia- 
stases on  fatty  matters.  The  diastases  acting  on  the  proteid 
substances  also  produce  a  decomposition  and  simultaneously 
a  hydration,  although  in  this  case  it  would  be  difficult  to 
demonstrate  the  reaction. 

The  molecules  of  albuminoid  substances  are  excessively 
complex ;  it  is  generally  assumed  that  they  have  a  molecular 
weight  of  about  5500,  and  as  certain  products  of  cleavage 
have  molecular  weights  of  2800,  of  1400,  and  of  400,  it  can 
be  seen  that  diastatic  action  causes  a  diminution  of  molecular 
weight. 

Enzymes  hydrating  or  decomposing  the  molecules  may 
give  rise  to  two  different  substances,  as  saccharose  yields 
dextrose  and  levulose. 

In  the  inversion  of  lactose,  one  observes  the  same 
phenomenon;  the  two  portions  into  which  the  molecules  are 
decomposed  are  different,  dextrose  and  galactose. 

There  are  also  cases  of  cleavage  in  which  two  molecules 
of  identical  chemical  configuration  are  produced.  Thus  the 
diastase  found  by  Cusenier,  glucase,  acts  on  maltose,  giving 
two  molecules  of  dextrose. 

BIBLIOGRAPHY. 

Kirchoff. — Formation  du  sucre  dans  les  cereales.     Journal  de  Pharmacie. 

1816,  p.  250.    Acad,  de  St.  Petersbourg,  1814. 
Dubrunfaut. — Memoire  sur  la  saccharification.     Societe  d'agriculture  de 

Paris,  1823. 
Payen  et  Persoz. — Memoire  sur  les  diastases  et  les  principaux  produits  de 

leur  action.     Ann.  de  chimie  et  de  phys.,  1833,  p.  73. 
Reaumur. — Memoire  sur  la  digestion  des  oiseaux.     Histoire  de  l'Acad. 

des  sciences,  1752,  p.  266-461. 


26  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

Spallanzani. — Experiences  sur  la  digestion.  Geneve,  1783. 
Schwann. — Essence  de  la  digestion.  Mullers  Archiv,  1836. 
Schaer. — Uber    die     Guajaktinktur-Reagentz.     Apoth.     Zeitung.     Berlin, 

1894- 
Jacobson. — Untersuchungen  uber  die  losliche  Fermente.     Zeitschrift  fur 

phys.   Chemie,  1892,   16,  p.  340-369. 
Dastre. — Solubilite  et  activite  des  ferments  solubles  en  liquide  alcoholique. 

Bull.  de.  l'Acad.  des  sciences,  1895,  24,  p.  899. 
Nikilta  Chodschajew. — Dialyse  des  enzymes.     Archiv  physiolog.,   1898. 
Bouchardot. — Sur    le.   ferment    saccharirhant    ou    glucosique.     Ann.     de 

chimie   et  de  physique,   1845. 
Muntz. — Sur   le  ferment   chimique   et  physiologique.     Comptes    Rendus, 

1875.     Ann.  de  chimie  et.de  physique,  5,  p.  428. 
JSTasse.und  Framm. — Glycolyse.     Pfliigers  Archiv,  63,  p.  203-208. 
J.   Raulin. — Recherches  sur  le  developpement  d'une  mucedinee  dans  tin 

milieU'artificiel.     Ann.  des  sciences. naturelles,  1890. 
Scruff. — Leqons    sur    la    digestion    stomacale,     1872-73.     Traduct.  franc. 

Paris,  1868. 
Effront.— Sur   les   conditions   chimiques   de   Taction    des    diastases,    1892. 

Comptes  Rendus  ded'Ac.  des  sciences,  p.  1324. 
Beijerinck. — Centralblatt. fiir  Bacteriologie,  II,  1898. 
"VVroblewsky. — Uber    die    chemische    Beschaffenheit    der    amylolytischen 

Fermente.     Berichte  der  deut.  chem.  Gesellschaft,  1898. 
Bourquelot. — Sur  les  caracteres  pouvant  servir  a  distinguer  la  pepsine  de 

la  trypsine.     Jour,  de  Pharmacie  et  Chimie,  1884. 
Wiirtz. — Sur  le   ferment  digestif   du   Carica.  papaya.     Comptes    Rendus, 

1879,  t.  XXXIX,  p.  425. 
Gautier. — Sur  les   modifications   solubles   et   insolubles   du  ferment  de  la 

digestion  gastrique.    Comptes  Rendus,  1892,  p.  682. 
Sur  la  modification  insoluble  de  la  pepsine.     Comptes  Rendus,  1892, 

p.  1192,  .t.  XCIV. 
A.     Wroblewsky. — Natur     der     Enzyme     Classification.     Berichte     der 

deutschen  chem.  Gesellschaft,  1897,  3,  30408. 
R.  Pawlewsky. — Unsicherheit  der  Guajakreaction.  Berichte  der  deutschen 

chem.  Gesellschaft,  1897,  2,  1313. 


CHAPTER  III. 

MANNER  OF  ACTION  OF  DIASTASES. 

Manner  of  action  of  diastases. — Different  opinions  on  this  subject. — The 
diastatic  property  and  the  diastase  itself. — Works  of  Bunzen,  Hiifner, 
Naegeli,  Wittich  and  Fick,  de  Jager,  Arthus. — Analogy  between  or- 
ganized ferments  and  soluble  ferments. — Hypothesis  of  Armand 
Gautier  on  the  nature  of  enzymes. 

We  have  seen  above  that  enzymes  can  produce,  according 
to  their  nature,  a  molecular  change,  a  hydration  or  an  oxida- 
tion. We  have  also  seen  that  diastatic  actions  are  charac- 
terized by  the  disproportion  existing  between  the  results  pro- 
duced and  the  weight  of  the  active  substance.  This  dispro- 
portion between  cause  and  effect  proves  that  the  active 
substances  do  not  enter  into  the  definite  composition  of  the 
products  whose  formation  they  induce.  Enzymes  appear  to 
us  to  play  in  these  transformations  the  part  of  intermediaries, 
provided  with  the  property  of  increasing  the  interior  energy 
of  the  substances  on  which  they  act  and  rendering  them  more 
liable  to  decompositions  or  combinations. 

Berzelius  has  compared  diastatic  reactions  to  the  phe- 
nomena called  catalytic  phenomena  which  were  formerly  ex- 
plained simply  by  the  effect  of  contact  or  the  presence  of  a 
body.  This  investigator  had  noticed  an  analogy  between 
the  action  produced.by  an  enzyme  and  the  decomposition  of 
hydrogen  peroxide  by  spongy  platinum. 

The  comparison  of  Berzelius  is  not  a  happy  one.  Hydro- 
gen peroxide  is  a  very  unstable  substance,  and  porous  bodies, 
as  powdered  charcoal,  and  metals  in  finely  divided  condition, 
cause  the  decomposition  of  certain  bodies  on  account  of  their 

27 


28  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

extreme  porosity.  Now,  it  is  evident  that  enzymes  do  not 
act  in  this  way,  and  the  relation  imagined  by  Berzelius  is  still 
more  strange  in  that  there  is  on  one  hand  a  reaction  between 
a  liquid  and  a  solid,  and  on  the  other  an  action  which  takes 
place  exclusively  between  bodies  in  solution.  But  if  the  ex- 
ample cited  by  Berzelius  is  ill-chosen,  it  must  still  be  acknowl- 
edged that  enzymes  appear,  at  first  sight,  to  act  by  simple 
contract,  and  that,  in  fact,  there  exists  a  striking  analogy  be- 
tween catalytic  reactions  and  diastatic  actions.  In  the  two 
cases  one  finds,  at  the  end  of  the  reaction,  the  body  acting  in 
the  same  state  as  at  the  beginning,  and  one  observes  that  the 
quantity  of  excitant  allowed  to  act  has  no  influence  on  the  re- 
sults obtained.  There  is  known  in  inorganic  and  in  organic 
chemistry  a  whole  series  of  reactions  of  that  nature :  the  de- 
composition of  calcium  hypochlorite  by  cobaltic  oxide,  of 
hydrogen  peroxide  by  potassium  bichromate,  the  combina- 
tion of  benzene  with  methyl  chloride  in  presence  of  alumin- 
ium chloride,  etc.  In  all  these,  the.  acting  substance  re- 
mains at  the  end  of  the  reaction:  It  is  phenomena  of  this 
kind  that  were  formerly  considered  as  catalytic  reactions,  but 
the  real  workings  of  which  are  now  known. 

It  is  thus  that  the  decomposition  of  calcium  hypochlorite 
by  certain  metallic  oxides,  by  cobaltic  oxide,  for  example,  ap- 
pears to  be  brought  about  by  the  simple*  presence  of  the 
cobaltic  oxide,  because  this  body  is  found  again  intact  and 
appears  to  have  undergone  no  change  during  the  reaction. 
But  in  reality  its  part  is  not  one  of  complete  indifference: 
there  is  formed  during  the  reaction  cobaltous  oxide,  which  is 
then  oxidized  and  which  can  act  anew  on.  the  hypochlorite. 

(i)       (C10)2Ca  +  2Co203  =  CaCl2  +  202  +  4C0O. 

Hypochlorite  Cobaltic  Cobaltous 

of  calcium.  oxide.  oxide. 

(2)  4C0O  +  02  =  2Co203. 

When  hydrogen  peroxide  has  been  decomposed  by  potas- 
sium bichromate,  we  have  an  analogous  process.     Potassium 


MANNER   OF  ACTION   OF  DIASTASES.  29 

bichromate  possesses  the  property  of  decomposing  little  by 
little  an  unlimited  amount  of  hydrogen  peroxide,  being  itself 
unchanged  at  the  end  of  the  reaction. 

Berthelot  explains  this  phenomenon  by  the  formation  of 
an  intermediate  compound,  repeatedly  destroyed  and  re- 
newed, and  whose  destruction  and  recomposition  are  carried 
on  until  the  moment  when  all  the  hydrogen  peroxide  is  de- 
composed. 

Berthelot,  by  adding  ammonia  to  a  mixture  of  hydrogen 
peroxide  and  dissolved  bichromate,  obtained,  when  the 
oxygen  was  liberated,  a  precipitate  composed  of  hydrogen 
peroxide,  and  chromic  oxide,  and  ammonium  chromate. 

It  is  the  compound  of  hydrogen  peroxide  with  chromic 
oxide  which,  during  the  reaction,  is  again  transformed  into 
chromic  acid  and  water.  The  reaction  probably  takes  place 
according  to  the  formula  : 

6H202  +  2Cr03  =  Cr203  .  3H202  +  3H0O  +  3O0. 
(Cr203  .  3H202)  =  2Cr03  +  3H20. 

In  organic  chemistry  one  can  obtain  reactions  entirely 
analogous.  It  is  thus  that,  in  the  reaction  of  Friedel  and 
Crafts,  metallic  salts  favor  the  substitution  of  monatomic 
groupings  for  atoms  of  hydrogen  in  the  benzene  series. 

Benzol,  C6H6  ,  and  methyl  chloride,  CH3C1,  do  not  act 
upon  each  other  under  ordinary  conditions ;  but  in  the  pres- 
ence of  a  metallic  salt,  such  as  aluminium  chloride,  there  is 
formed  toluol,  CGH5-CH3  ,  and  hydrochloric  acid,  HC1.  The 
function  of  the  salt  is  to  form  an  intermediate  combination 
which  facilitates  the  reaction. 

C6H6  +  Al2Clfi  =  C6H5A12C15  +  HC1. 
C6H5A12C15  +  CH3C1  =  C6H5-CH3  +  A12C16. 

On  the  whole,  all  these  reactions  resemble  each  other  in 
principle;  and  the  formation  of  ether  by  the  action  of  sul- 
phuric acid  upon  alcohol  may  serve  as  a  characteristic  ex- 
ample of  this  kind  of  reaction.  The  transformation  of  alcohol 


3°  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

is  produced  in  two  phases :  in  the  first  stage  the  alcohol 
combines  with  the  sulphuric  acid  to  form  ethyl  sulphuric  acid ; 
in  the  second  stage  the  product  formed  acts  again  on  the 
alcohol:  ether  is  formed  and  the  sulphuric  acid  is  regen- 
erated. 

It  is  very  probable  that  the  molecules  of  enzymes  form 
with  substances  subject  to  their  action  combinations  which 
are  not  permanent,  unstable,  and  which  are  easily  decom- 
posed, either  by  water  or  by  oxygen.  This  theory  may 
be  presented  in  the  following  manner.  Two  bodies  hav- 
ing feeble  affinities,  as  for  example  starch  and  water, 
are  made  to  react  by  the  aid  of  a  third  substance, 
for  example  malt  diastase.  A  molecular  combination  of  the 
starch  with  the  diastase  is  thus  secured.  This  combination 
has  no  longer  the  properties  of  the  bodies  which  have  entered 
into  its  composition;  it  is  a  substance  much  less  stable  which 
is  decomposed  by  water.  Following  this  decomposition  the 
diastase  reappears  in  its  original  state,  the  water  remaining 
combined  with  the  starch  molecule,  and  this  hydration  trans- 
forms the  starch  into  sugar.  This  theory,  due  to  Bunzen  and 
to  Hufner,  is  unfortunately  based,  not  on  precise  facts,  but 
on  analogies,  and  this  circumstance  renders  the  theory  open 
to  question. 

Wiirtz  attempted  to  prove  this  hypothesis  by  studying 
papain,  which  is  known  to  be  an  enzyme  acting  on  albuminoid 
substances.  He  observed  that  fibrin  immersed  in  a  solution 
of  this  enzyme  united  with  the  active  substance  in  such  a  way 
that  the  fibrin  could  be  washed  without  losing  it.  The  albu- 
minoid substance  thus  impregnated  was  transformed,  lique- 
fied, and  peptonized  as  soon  as  it  was  brought  to  a  tempera- 
ture favorable  to  diastatic  action.  As  the  same  phenomenon 
is  observed  with  pepsin,  Wiirtz  considered  that  albuminoid 
substances  form  with  enzymes  insoluble  combinations,  and 
that  these  combinations  are  the  intermediate  stages  analo- 
gous to  those  found  in  all  catalytic  reactions. 

Unfortunately  this  explanation  is  far  from  being  accurate, 


MANNER   OF  ACTION   OF  DIASTASES.  31 

for  the  diastases  are  fixed  not  only  on  the  bodies  which  they 
are  capable  of  transforming,  but  also  on  bodies  upon  which 
they  have  no  action,  as  silk.  From  another  point  of  view 
albuminoid  substances  form  combinations  analogous  to 
those  which  have  just  been  cited,  not  only  with  the  diastases 
which  are  able  to  act  upon  them,  but  also  with  other  enzymes 
incapable  of  transforming  them.  Thus,  the  experiments  of 
YViirtz  do  not  at  all  prove  the  formation  of  intermediate 
bodies.  Still  his  theory  of  the  manner  of  action  of  diastases 
finds  support  in  the  experiments  of  Schoenbein,  Schaer,  and 
Bikhner  relative  to  the  action  of  hydrocyanic  acid  on  active 
substances. 

Hydrocyanic  acid  added  to  an  aqueous  solution  of  dia- 
stase prevents  the  latter  from  decomposing  hydrogen 
peroxide  and  from  transforming  bodies  on  which  the  dia- 
stase can  act.  Still  hydrocyanic  acid  does  not  destroy  the 
diastase ;  in  fact,  when  a  current  of  air  has  been  passed 
through  the  inactive  solution,  the  activity  of  the  enzyme  re- 
appears. One  may  conclude  that  the  enzymes  form  with  the 
hydrocyanic  acid  an  unstable  combination  which  is  destroyed 
by  the  passage  of  a  current  of  air. 

These  views  are  very  favorable  to  the  theory  of  Bunzen. 
If  it  is  once  established  that  the  enzymes  can  form  inter- 
mediate combinations  with  hydrocyanic  acid,  it  may  also  be 
admitted  that  they  react  with  the  substances  which  they 
affect,  and  form  combinations  of  the  same  nature.  These  in- 
termediate substances  furnished  by  the 'hydrocyanic  acid  un- 
fortunately cannot  be  isolated  in  a  pure  state,  and  the  hy- 
pothesis of  Wiirtz,  although  probable,  is  not,  however,  based 
upon  rigidly  demonstrated  facts.  It  is  then  quite  natural  to 
seek  to  explain  the  manner  of  action  of  enzymes  by  other 
hypotheses. 

Xaegeli  explains  the  action  of  enzymes  in  an  entirely  .dif- 
ferent manner;  he  does  not  consider  the  action  of  diastases 
as  a  purely  chemical  phenomenon,  but  as  being,  at  least  par- 
tially, of  a  physical  nature.     This  investigator  considered 


32  THE  ENZYMES   AND    THEIR   APPLICATIONS. 

that  the  molecules  of  the  enzymes  are  animated  by  special 
vibrations  capable  of  producing  in  the  fermentable  substance 
molecular  vibrations  which  can  break  down  the  molecules. 
As  may  be  seen,  this  theory  greatly  resembles  the  old  theory 
of  fermentation  of  Liebig,  according  to  whom  the  phenomena 
of  fermentation*  in  general  are  brought  about  by  substances 
in  process  of  decomposition,  which  communicate  to  other 
bodies  in  contact  with  them  the  same  molecular  movement. 

This  hypothesis,  based  on  very  speculative  considera- 
tions, was  finally  taken  up  again  by  de  Jager.  He  carried  his 
conclusions  much  further,  and  thought  to  bring  out  facts 
demonstrating  that  enzymes  act,  not  as  substances,  but 
rather  as  forces.  The  experiments  on  which  de  Jager  based 
his  views  are  due  to  Wittich  and  Fick. 

Fick  placed  a  solution  of  rennin  in  glycerin  in  a  long 
tube,  then  carefully  filled  it  with  milk.  As  the  rennin  did  not 
diffuse,  de  Jager  concluded  that  the  coagulation  was  pro- 
duced, not  by  the  rennin,  but  by  a  property  inherent 'in  that 
substance. 

Wittich  placed  in  a  dialyser  furnished  at  its  lower  base 
with  a  parchment  membrane,  some  pepsin  dissolved  in  a  cer- 
tain quantity  of  water.  He  then  introduced  this  dialyser  into 
a  larger  basin  containing  water  and  flakes  of  fibrin.  He  did 
not  detect  any  dialysis  of  the  pepsin,  yet  the  flakes  of  fibrin 
liquefied  and  became  peptonized  as  if  they  had  been  in  con- 
tact with  the  enzyme. 

It  is  very  evident  that  if  these  experiments  were  exact, 
the  theory  that  enzymes  act  as  chemical  substances  would 
be  discredited,  and  it  would  then  appear  that  the  action  of 
enzymes  must  be  purely  physical.  But  other  investigators 
who  have  repeated  these  experiments  have  never  been  able 
to  reach  the  results  described. 

The  opinion  of  de  Jager  has  been  quite  recently  taken  up 
by  Arthus  who,  while  recognizing  the  inaccuracy  of  the  ex- 
periments of  Fick  and  Wittich,  still  inclines  to  support  the 
theory  of  enzyme-properties. 


MANNER   OF  ACTION   OF  DIASTASES.  33 

Arthus  has  been  no  more  successful  than  his  predecessois 
in  performing  experiments  which  decisively  favor  his  theory, 
but  he  shows  effectively  the  weaknesses  of  the  theory  which 
considers  enzymes  as  substances.  He  first  notes  that  the 
percentage  analysis  is  not  enough  to  characterize  enzymes. 
He  emphasizes,  as  we  have  done  above,  the  disagreement 
existing  between  the  analyses  of  enzymes  made  by  different 
authors ;  he  shows  also  that  diastases  cannot  be  classed  in 
any  definite  chemical  group,  for  they  are  neither  albuminoid 
substances  nor  gums.  He  is  especially  struck  with  the  fact 
that  each  author  claims  to  have  prepared  a  pure  enzyme  by 
successive  precipitations,  without  one  of  them  being  able  to 
say  by  what  characteristics  a  pure  enzyme  is  to  be  recog- 
nized. He  has  also  remarked  that  enzymes  appear  to  vary 
in  composition  and  properties  according  to  the  manner  in 
which  they  have  been  prepared.  He  then  cites  the  opinions 
of  various  authors  on  the  impurity  of  diastatic  precipitates, 
and  concludes  that  all  diastases  which  have  been  analyzed  up 
to  the  present  time  were  much  mixed  with  foreign  sub- 
stances. He  shows  himself  equally  opposed  to  the  theory 
which  seeks  to  show  an  analogy  between  the  manner  of 
formation  of  ether  by  sulphuric  acid  and  the  action  of 
enzymes.  He  bases  this  opinion  on  the  difference  between 
the  quantity  of  sulphuric  acid  necessary  to  transform  the 
alcohol  into  ether,  and  the  quantity  of  enzyme  which  must  be 
used  to  work  a  diastatic  transformation.  In  fact,  sulphuric 
acid  etherizes  only  25  to  30  times  its  weight  of  alcohol,  while 
a  certain  quantity  of  diastase  transforms  quantities  of  matter 
infinitely  great  in  relation  to  itself.  Rennin,  for  example,  can 
coagulate  250,000  times  its  weight  of  casein.  He  shows 
finally  that  the  properties  of  enzymes  do  not  at  all  necessitate 
that  these  latter  shall  be  chemical  substances,  but  that  they 
may  be  imponderable  agents,  like  heat,  electricity,  etc. 

To  demonstrate  this,  Arthus  takes  up  the  properties 
of  diastases  one  by  one,  and  attempts  to  match  with  them 
analogous     phenomena     of     light,     heat,     and     electricity. 


34  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

Enzymes  bring  about  chemical  transformations;  but  light, 
heat,  and  electricity  also  bring  them  about:  of  this,  elec- 
trolysis is  a  striking  example.  Enzymes  are  destroyed  by 
heat ;  in  like  manner,  a  magnetized  bar  loses  its  magnetic 
property  when  it  is  heated  red-hot.  Enzymes  are  soluble  in 
water  and  glycerin;  but  when  a  warm  body  is  plunged  in 
any  liquid  whatever,  the  latter  is  heated,  although  the  body 
may  not  dissolve.  Enzymes  are  thrown  down  from  their 
solutions  by  alcohol  or  by  mechanical  precipitants ;  but 
sodium  chloride  precipitated  by  alcohol  also  stores  up  a  cer- 
tain quantity  of  heat  which  reappears  when  it  is  redissolved 
in  water.  In  the  same  way,  diastatic  precipitates  thrown 
down  by  alcohol  enclose  a  certain  quantity  of  the  enzyme, 
and  this  enzyme  reappears  when  placed  in  a  suitable  medium. 
Enzymes  are  retained  by  fresh  fibrin ;  but  electric  accumula- 
tors retain  electricity,  and  certain  bodies,  as  barium  sulphide, 
absorb  rays  of  light.  Certain  substances,  under  the  action 
of  chemical  agents,  acquire  a  diastatic  power;  so  the  oxides 
of  phosphorus  are  known  to  diffuse  light.  Enzymes  are  de- 
stroyed by  certain  agents ;  the  magnetism  of  a  magnetized 
bar  disappears  when  the  bar  is  dissolved  in  hydrochloric  acid. 
The  action  of  diastases  is  hindered  by  certain  bodies  and 
facilitated  by  others;  in  an  electric  current,  if  a  resistance  is 
interposed,  the  intensity  of  the  current  diminishes,  and  if,  on 
the  contrary,  this  resistance  is  removed,  the  current  increases 
in  intensity.  Diastatic  action  is  generally  produced  on  cer- 
tain bodies  to  the  exclusion  of  others ;  iron  and  steel  alone 
can  fix  the  magnetic  property. 

Arthus  concludes  from  all  these  comparisons  that  en- 
zymes are  not  substances  but  properties  of  substances.  He 
admits  that  his  theory  has  not  been  demonstrated,  but  on  the 
other  hand,  he  asserts  that  the  theory  of  enzyme-substance 
has  not  received  any  clearer  demonstration. 

On  the  whole,  we  recognize  the  existence  of  two  theories. 
The  one  assumes  that  enzymes  act  chemically  and  that  they 
have  a  definite  chemical  composition,  the  other  considers  the 


MANNER   OF  ACTION  OF  DIASTASES.'  35 

enzyme  as  a  property  and  not  as  a  substance.  The  argu- 
ments which  Arthus  brings  up  against  the  theory  of  en- 
zyme-substances are  not  of  a  nature  to  overthrow  that  theory. 
The  disagreement  between  the  analyses  of  the  same  diastase 
may  very  well  be  due  to  the  manner  of  preparation  and  puri- 
fication of  that  substance.  If  it  could  be  proved,  on  the  other 
hand,  that  diastases  cannot  be  classified  in  any  chemical 
group  actually  known,  that  would  not  disprove  the  existence 
of  diastases  as  substances.  In  fact,  at  the  present  time  we  are 
still  far  from  knowing  all  the  chemical  combinations,  and  it 
is  more  than  probable  that  there  exists  a  great  number  of 
bodies  about  which  we  know  nothing.  The  fact  that  enzymes 
act  in  infinitely  small  amounts  is  not  at  all  of  a  nature  to  in- 
validate the  hypothesis  of  enzyme-substance.  In  the  action 
of  strychnin,  aconite,  and  many  other  alkaloids,  there  is  seen 
also  an  enormous  disproportion  between  the  result  produced 
and  the  weight  of  the  acting  substance.  The  action  of  musk 
is  indisputably  much  more  sensitive  than  the  action  of 
enzymes :  reactions  can  be  obtained  on  the  olfactory  nerve- 
endings  with  exceedingly  small  amounts,  and  this  remark- 
able property  is  entirely  due  to  the  chemical  constitution  of 
these  bodies. 

The  parallel  between  the  phenomenon  of  fermentation 
and  physical  phenomena  is  very  attractive  ;  but,  on  the  whole, 
the  hypothesis  of  enzymes,  as  properties,  is  much  less  prob- 
able than  that  of  enzymes  as  substances.  One  always  finds 
the  capacity  for  action  incorporated  in  a  material  substance, 
and  one  has  never  succeeded  in  separating  the  property  from 
the  substance.  There  is  nothing  to  justify  a  belief  that 
material  substance  plays  no  part  in  the  diastatic  phe- 
nomenon. 

Enzymes  present  various  resemblances  to  living 
protoplasm. 

The  enzyme,  like  the  living  organic  substance,  is  ex- 
tremely sensitive  to  chemical  agents,  such  as  acids  and  alka- 
li' :s,     These  two  classes  of  substances  are  destroyed  at  a  tern- 


36  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

perature  of  ioo°,  and  they  have  the  property  of  exciting- 
chemical  reactions  in  the  surrounding  medium. 

Most  enzymes  have  a  chemical  composition  very  similar 
to  that  of  protoplasm,  and  both  these  materials  furnish  some 
reactions  common  to  albuminoid  substances.  The  analogy 
becomes  still  more  striking  when  one  studies  the  composi- 
tion of  the  mineral  substances  which  evidently  enter  into  the 
chemical  composition  of  protoplasm  and  soluble  ferments. 
Both  contain  phosphates  of  calcium,  potassium,  and  mag- 
nesium, and  alkaline  chlorides  and  sulphates.  The  mineral 
and  organic  elements  which  are  favorable  to  the  living  cell 
are  also  exciting  agents  for  certain  diastases,  as  has  been 
demonstrated  for  asparagin  and  phosphates.  Enzymes,  like 
protoplasmic  substance,  are  diffusible  with  difficulty,  and  in 
many  cases  do  not  even  pass  through  a  porcelain  filter. 
One  may  then  consider  that  diastases  are  not  even  soluble 
bodies,  properly  speaking,  and  that  when  they  are  in  contact 
•with  water  they  go  only  into  a  state  of  extreme  tenuity,  as 
do  colloid  bodies  such  as  starch-powder. 

This  analogy  between-organic  substance  and  enzymes  has 
led  Armand  Gautier  to  suppose  that  chemical  ferments  re- 
semble in  their'  constitution  the  cells  from  which  they  are 
derived.  He  considers  further,  that  enzymes  have  an  or- 
ganization analogous  or  very  similar  to  that  of  protoplasm, 
and  he  credits  them  with  the  fundamental  properties  of  the 
living  cell,  which  are  assimilation  and  reproduction.  Ac- 
cording to  this  investigator,  enzymes  can  transform  certain 
substances  into  bodies  like  themselves.  On  the  strength  of 
this  bold  hypothesis,  he  cites  a  single  experiment,  made  with 
pepsin,  and  to  which  we  shall  have  occasion  to  return  when 
we  come  to  study  the  action  of  enzymes  upon  proteid  sub- 
stances. 

For  the  present  we  may  say  that  the  experiment  cited  by 
Gautier  does  not  at  all  confirm  his  theory,  and  we  are 
rather  inclined  to  regard  enzymes  as  chemical  bodies  of  a 
particular  nature  and  a  definite  constitution.     And  in  reality, 


MANNER   OF  ACTION   OF  DIASTASES.  37 

in  proportion  as  our  knowledge  of  diastases  increases,  the 
theory  of  enzyme-substance  seems  more  and  more  probable. 
At  present  we  possess  an  array  of  facts  which  indicate  that 
we  are  really  dealing  with  bodies  and  not  with  properties. 
We  know,  for  example,  that  amylase  procured  from  various 
media,  from  raw  grain,  from  malted  grain,  from  the  saliva, 
from  the  pancreatic  juice,  from  bacteria  and  moulds,  always 
shows  the  same  chemical  composition  and  always  gives  the 
reaction  of  a  proteose. 

The  chemical  nature  of  enzymes  is  still  further  confirmed 
by  the  color  reactions  which  they  show  with  certain  reagents. 
As  Guignard  has  shown,  emulsin  gives  a  violet  color  with 
orcin  and  a  red  color  with  Millon's  reagent.  Another 
enzyme,    myrosin,  gives  a  violet  tint  in  hydrochloric  acid. 

In  special  cases  it  has  been  possible  to  cause  one  enzyme 
to  react  upon  another.  This  action  is  very  characteristic 
and  affords  data  on  the  chemical  nature  of  enzymes.  Ac- 
cording to  Naegeli  and  Kiihne  pepsin,  for  example,  acts 
upon  trypsin  as  upon  an  albuminoid  substance.  Chittenden 
and  Griswold  have  observed  the  same  phenomenon  with 
ptyalin:  this  enzyme  is  also  attacked  by  pepsin.  The  zymase, 
or  enzyme  producing  the  alcoholic  fermentation,  is 
destroyed,  according  to  Biichner,  in  the  presence  of 
trypsin.  In  the  action  of  one  enzyme  upon  another,  one  of 
the  active  substances  is  always  hydrated  and  chemically 
changed,  with  the  complete  suppression  of  its  activity. 

As  pepsin  and  trypsin  act  exclusively  upon  albuminoid 
substances,  one  may  conclude  that  ptyalin  and  trypsin  as  well 
as  zymase  belong  to  that  class  of  substances. 

The  existence  of  enzymes  containing  little  or  no  nitrogen 
cannot,  however,  constitute  an  argument  for  the  support  of 
the  theory  of  enzyme-properties.  Different  diastases  act  on 
different  bodies,  and  cause  very  varied  reactions.  It  is  then 
very  evident  that  all  active  substances  cannot  belong  to  the 
same  class  of  bodies  and  that,  in  all  probability,  the  enzymes 
will  present  wide  differences  in  composition  and  structure. 


38  THE  ENZYMES  AND    THEIR  APPLICATIONS. 


BIBLIOGRAPHY. 

Liebig. — Sur  les  phenomenes  de  la  fermentation  et  de  la  putrefaction  et 

sur  les  causes  qui  les  provoquent.     Ann.   de  chimie  et  de   physique, 

1839,  t.  LXXI,  p.  147. 
Wiirtz. — Sur  la  papa'ine,  contribution  a  l'histoire  des  ferments  solubles. 

Comptes   Rendus,    1880,   p.    1379. 
Sur  la  papa'ine,  nouvelle  contribution  a  l'histoire  des  ferments  solu- 
bles.    Comptes   Rendus,   1880,  p.  787. 
Wiirtz  et  Bouchut. — Sur  le  ferment  digestif  du  Carica' papaya.     Comptes 

Rendus,  1889,  p.  425. 
O.  Loew. — Ueber  die  Natur  der  ungeformten  Fermente.    Pfliigers  Archiv 

fur  die  gesammte  Physiol.,   1885,  Band  36,  p.   170. 

Pfliigers  Archiv,  1881,  p.  205. 

C.  J.  Lintner. — Ueber  die  chemische  Natur  der  vegetabilischen  Diastase. 

Bemerkungen    zu    der   Arbeit    Herschlegers.     Pfliigers    Arch.,    1887, 

Band  49,  p.  311. 
E.  Hirschfeld. — Ueber  die  chemische  Natur  der  vegetabilischen  Diastase. 

Pfliigers  Archiv  fur  die  gesammte  Physiologie,  1886,  Band  39,  p.  499. 
Latschenberger. — Ueber     die     Wirkungsweise     der     Gahrungsfermente. 

Centralblatt  fur  Physiol.,  1891,  Band  IV,  p.  3. 
Huefner. — Untersuchungen  iiber  die  ungeformten  Fermente.     Jour,  fur 

prakt.  Chemie,  B.  V,  p.  372. 
■ Recherche   sur   le   ferment   non   organise.     Bull,    de   la   Soc.    chim. 

Paris,  1877. 
Jacobson. — Untersuchungen  iiber  losliche   Fermente.     Zeit.  fur  physiol. 

Chem.,  XVI,  p.  340. 
L.  de  Jager. — Erklarungsversuch  iiber  die  Wirkungsart  der  ungeformten 

Fermente.    Virchows  Archiv,  Band  121,  1890,  p.  182. 
V.  Wittich. — Weitere  Mittheilungen  iiber  Verdauungsfermente  des  Pep- 
sines  und   seine  Wirkung  auf  Blutfibrin.     Pfliigers   Arch.,    Band   5, 

p.  43s,   1872. 
A.    Fick. — Ueber    die    Wirkungsart    der    Gerinnungsfermente.     Pfliigers 

Arch.,  Band  45,  sect.  293,  1889. 
Effront. — Comparaison  entre  le  role  des  Diastases  et  celui  de  la  nutrition 

minerale.    Moniteur  scientifique,  1894. 
Maurice  Arthus. — Nature  des  enzymes.     These  pour  le  doct.  en  medec. 

Paris,   1896. 
Schoenbein. — Ueber  das  Verhalten  der  Blausaure  zu  den  Blutkcrp;rch  n 

und    den   iibrigen    organischen    das    H2O2   katalysirenden    Mater.en. 

Zeit.  fur  Biologie,  Band  111,  p.  140. 
Ed.  Schaer. — Der  thatige  Sauerstoff  und  seine  physiologische  Bedeutung. 

Wittsteins  J.  fur  praktische  Pharmacie,  1869,  III,  IV. 
Beitrage  zur  Chemie  des  Blutes  und  der  Fermente.     Zeit.  fur  Bio- 
logie, 1870,  p.  467. 


MANNER   OF  ACTION  OF  DIASTASES.  39 

Ed.  Schaer. — Ueber  den  Einfluss  des  Cyanwasserstoffs  und  Phenols  auf 

gevvisse  Eigenschaften  der  Blutkorperchen  und  Fermente. 
Biichner. — Berichte    der    deutschen    chemisch.    Gesellschaft,     1897,    No. 

2608. 
Kahn. — Berichte  der  deutschen  chemischen  Gesellschaft,  1898. 
A.  Gautier. — Les  toxines  microbiennes  et  animales.     Paris  Soc.  d'editions 

scientifiques. 
G.    Tammann. — Zur    Wirkung    ungeformter    Fermente.     Zeitschrift    fiir 

phys.  Chemie,  p.  421,  442. 


CHAPTER  IV. 

INDIVIDUALITY  OF  ENZYMES. 

Difficulties  encountered  in  proving  the  individuality  of  enzymes. — Influ- 
ence of  the  nutrition  of  cells  upon  the  nature  of  enzymes  which  they 
secrete. — Direct  proofs  of  the  individuality  of  enzymes. — Relation 
among  the  diastases;  chemical  constitution  and  structure  of  the 
bodies  upon  which  they  act. — Nomenclature  of  enzymes. — Classifica- 
tion. 

When  one  studies  the  active  substances  secreted  by  living 
cells  with  reference  to  their  chemical  action,  one  finds  gener- 
ally that  this  action  is  very  complex,  that  different  substances 
are  affected,  and  that  very  varied  products  arise.  Thus,  an 
infusion  of  malt  acts  upon  starch,  cellulose,  pectin,  trehalose, 
and  caroubin.  Furthermore,  the  results  obtained  with  these 
various  substances  are  very  different :  The  diastase  in  acting 
on  starch  gives  maltose  and  dextrins,  it  liquefies  the  cellulose, 
transforms  the  pectic  materials  into  a  gelatinous  substance, 
causes  the  trehalose  to  pass  into  monosaccharid,  and 
changes  the  caroubin  into  another  monosaccharid. 

We  observe  the  same  phenomenon  in  studying  the  prop- 
erties of  an  aqueous  extract  of  beer-yeast.  This  infusion 
acts  on  cane-sugar,  maltose,  and  the  glucosides,  and  gives  in 
every  case  a  specific  product. 

These  facts  lead  us  to  inquire  whether  living  cells  secrete 
a  single  active  substance  having  the  power  of  acting  on  dif- 
ferent chemical  combinations,  or  if,  on  the  contrary,  they 
give  rise  to  a  mixture  of  many  enzymes,  each  adapted  to  pro- 
duce a  special  action.  The  same  question  arises  for  enzymes 
precipitated  from  their  solutions,  for  they  also  generally  give 
very  varied  results. 

40 


INDIVIDUALITY  OF  ENZYMES.  4* 

It  is  difficult  to  answer  this  question  clearly  in  each  par- 
ticular case ;  but  in  general  the  individuality  of  enzymes  can- 
not be  denied. 

This  individuality  becomes  evident  in  many  cases,  when 
we  compare  the  actions  of  the  products  secreted  by  cells  of  a 
special  kind  placed  under  different  nutritive  conditions. 

Duclaux  has  found  that  by  cultivating  Pcnicillium  glaucum 
on  starchy  substances,  there  is  produced  in  the  culture 
medium  a  complex  substance  acting  on  cane-sugar  as 
well  as  on  starch.  It  is  very  difficult  to  give  a  direct  proof  of 
the  existence  in  this  medium  of  two  different  enzymes,  one 
acting  on  saccharose,  the  other  on  starch,  for  in  isolating  the 
active  substances  from  this  medium,  either  by  mechanical 
precipitation  or  by  alcohol,  one  obtains  still  another  sub- 
stance acting  upon  different  carbohydrates  and  giving  dif- 
ferent products. 

But  the  question  may  be  answered  by  making  a  second 
culture  of  Pcnicillium  glaucum,  and  in  the  culture  medium  re- 
placing the  starch  by  calcium  lactate.  The  active  substance 
which  is  formed  this  time  acts  very  strongly  on  the  cane- 
sugar  but  not  upon  starch.  We  may  then  conclude  that  in 
the  first  culture  there  were  present  two  enzymes,  while  in 
cultivating  the  mould  on  calcium  lactate  only  one  of  these 
was  obtained. 

Our  conclusion  will  be  strengthened  if  we  can  find  other 
examples  in  which  an  active  substance  will  produce  an  action 
exclusively  on  saccharose  or  on  starch.  These  examples  are 
very  numerous.  Thus  it  is  that  an  infusion  of  barley  acts  on 
starch  and  not  on  cane-sugar,  and  that  the  amylase  obtained 
from  the  rennet  of  sheep  acts  in  a  similar  manner.  One  may 
also  find  present  in  the  saliva  a  ferment  acting  on  the  starch 
without  causing  any  transformation  of  saccharose,  especially 
if  the  saliva  does  not  contain  any  organized  ferments.  An 
infusion  of  yeast  can  act  on  sugar  and  leave  starch  un- 
affected. 

In  certain  cases  the  individuality  of  enzymes  can  be  con- 


42  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

trolled  by  different  means  from  those  which  we  have  just  set 
forth.  If,  for  example,  we  leave  beer-yeast  in  prolonged  con- 
tact with  water  with  the  addition  of  ether  or  thymol,  we  find 
that  the  solution  acts  at  the  same  time  on  cane-sugar  and  on 
maltose.  We  may  then  ask  if  the  diastase  which  transforms 
the  maltose  does  not  also  act  on  cane-sugar.  It  is  easy  to 
demonstrate  here  the  existence  of  two  ferments.  It  is  only 
necessary  to  leave  the  yeast  in  contact  with  water  for  a  very 
•short  time  in  order  to  find  in  the  liquid  a  substance  acting 
on  cane-sugar  without  having  the  least  action  on  maltose. 
In  this  case  we  have  actually  separated  the  two  enzymes, 
owing  to  the  fact  that  one  of  them  is  not  firmly  held  by  the 
cells  which  secrete  it,  while  the  other  escapes  from  the  cells 
wTith  much  difficulty. 

We  have  seen  that  malt  diastase  acts  on  starch,  giving 
maltose  and  dextrins ;  it  also  acts  on  trehalose,  which  it  trans- 
forms into  a  monosaccharid.  Some  authors  have  concluded 
from  this  that  there  is  present  a  single  substance.  But  the 
amylase  which  is  taken  from  the  saliva  acts  upon  starch  in 
-exactly  the  same  way  as  the  amylase  from  malt,  while  it  has 
no  action  on  trehalose.  It  seems  to  us  that  in  this  case  also 
it  is  more  logical  to  assume  the  presence  of  two  different  en- 
zymes in  the  infusion  of  malt,  than  to  explain  the  phenom- 
enon by  supposing  that  there  exists  in  the  saliva  and  in  the 
malt  infusion  two  different  diastases,  both  acting  in  the  same 
manner  on  the  starch  and  differing  in  their  action  on 
trehalose. 

Emulsin  acts  on  glucosides,  but  at  the  same  time 
this  enzyme  will  transform  sugar  of  milk  into  dextrose  and 
-galactose.  In  emulsin  the  presence  of  two  diastases  be- 
comes evident,  in  our  opinion,  if  one  considers  that  the  active 
substances  secreted  by  certain  yeasts  have  the  power  of 
transforming  lactose  without  acting  in  any  way  on  glucosides. 

From  many  facts  of  this  kind  we  are  justified  in  conclud- 
ing that  in  a  majority  of  cases  cellular  secretions  are  com- 
posed of  different  active  substances,  and  that  the  chemical 


INDIVIDUALITY   OF  ENZYMES.  4,> 

action  of  each  of  these  enzymes  is  limited  to  a  certain  number 
of  bodies.  These  facts  are  sufficiently  numerous  for  us  to 
draw  a  general  conclusion  in  favor  of  the  individuality  of 
enzymes.  And  as  a  matter  of  fact,  it  is  difficult  to  believe 
that  the  same  active  substance  can  in  one  special  case  act  on 
two  or  three  chemical  substances,  while  in  another  case  its 
action  is  limited  to  a  single  one  of  these  substances. 

As  we  have  just  seen,  a  diastase  having  a  hydrating  or 
oxidizing  function  does  not  act  on  all  the  substances  capable 
of  being  hydrated  or  oxidized ;  the  diastatic  agent  differs 
completely  from  a  chemical  agent  having  a  definite  function 
and  exercising  it  independently  of  the  constitution  of  the 
bodies  on  which  it  acts.  For  example,  by  the  action  of  a 
mineral  acid  is  obtained  the  splitting  of  saccharose,  the 
saponification  of  fatty  matters,  the  decomposition  of  gluco- 
sides,  the  peptonization  of  albuminoid  substances,  in  a  word 
all  the  phenomena  which  we  meet  in  diastatic  hydrations. 
Among  diastases,  on  the  contrary,  decompositions  and  hy- 
drations are  caused  by  numerous  agents,  each  capable  of  a 
special  diastatic  work  and  acting  upon  only  a  very  limited 
number  of  substances.  The  action  of  acids  is,  then,  up  to  a 
certain  point,  independent  of  the  constitution  of  the  bodies 
on  which  they  act,  while  diastases  exercise  their  hvdrating  or 
oxidizing  action  only  on  bodies  of  a  strictly  definite  struc- 
ture. 

A  hydrating  enzyme  may  sometimes  exert  its  action  on 
different  bodies,  but  only  when  the  chemical  constitution  of 
these  bodies  is  very  much  like  that  of  the  diastase,  and  when 
they  can  furnish  the  same  products  of  decomposition.  It  is 
thus  that  amylase  acts  on  starch,  glycogen,  and  dextrin,  giv- 
ing always  the  same  end-product,  maltose. 

Pepsin  acts  on  a  great  number  of  bodies,  for  example,  on 
all  albuminoid  substances. 

Xow  all  these  bodies  resemble  each  other  and  have  a  very 
similar  structure,  since  their  products  of  decomposition  by 
the  diastase  are  always  the  same:  proteoses  and  peptones. 

The  enzymes  of  glucosides  appear  capable,  at  first  sight, 


44 


THE  ENZYMES  AND    THEIR  APPLICATIONS. 


of  a  more  energetic  action,  and  one  extending  to  chemically 
different  bodies,  but  this  anomaly  is  only  apparent :  emulsin, 
-which  acts  on  very  complex  bodies,  affects  only  that  part 
common  to  all  the  molecules  of  glucosides.  The  action  of 
emulsin  is  due  to  the  affinity  it  has  for  glucose,  and,  as  Emil 
Fischer  has  demonstrated,  this  affinity  is  explained  by  the 
stereochemical  structure  of  the  carbohydrate  molecules. 
Emulsin  acts,  not  only  on  natural  glucosides,  but  also  on 
artificial  ethers,  which  are  obtained  with  glucose. 

In  studying  the  action  of  enzymes  on  artificial  ethers, 
Emil  Fischer  has  found  this  very  interesting  fact :  that  the  ac- 
tion or  inaction  of  an  enzyme  depends,  not  only  on  the  com- 
position of  the  substance  on  which  it  is  made  to  act,  but  also 
on  its  configuration.  By  treating  glucose  with  methyl 
alcohol  in  the  presence  of  hydrochloric  acid  one  obtains  two 
isomeric  ethers,  differing  in  their  geometrical  structure  on 
account  of  the  asymmetrical  carbon  atoms  of  the  glucoside 
chain. 

The  formation  of  two  isomeric  ethers  is  easy  to  explain : 
the  aldehydic  glucose  group  disappears  by  the  action  of  the 
alcohol  in  presence  of  hydrochloric  acid,  and  dehydration  is 
produced  in  the  glucose  chain,  giving  rise  to  an  intra- 
molecular ethereal  group.  The  carbon  of  the  aldehydic 
group  thus  becomes  asymmetrical,  and  in  consequence  the 
appearance  of  two  stereo-isomers  becomes  comprehensible. 

The  two  isomeric  glucosides 


H-C-O-CH, 


HCOH 


HCOH 


CH 


HCOH 


CH.-C-O-H 


HCOH 


\  HCOH 


CH 


HOCH 


H2COH 


HOCH0 


a.  Methyl  dextroglucoside.  /5.  Methyl  dextroglucoside. 

behave  differently  under  the  action  of  enzymes. 


INDIVIDUALITY   OF  ENZYMES.  45 

Emulsin,  which  acts  on  certain  derivatives  of  dextrose 
and  galactose,  acts  also  on  the  /3-methyl  glucoside,  but  has 
no  action  on  the  isomer  a. 

In  the  beer-yeasts  is  found  another  soluble  ferment  which 
acts  on  natural  glucosides,  but  this  ferment  has  absolutely  no 
effect  on  the  /?-methyl  glucoside,  while  it  acts  on  the  isomer 
a. 

This  example  is  another  proof  of  the  individuality  of  en- 
zymes and  shows  in  a  striking  way  the  influence  which  the 
chemical  structure  of  bodies  has  on  the  diastatic  action. 

Emil  Fischer  has  evolved  the  hypothesis  that  a  diastatic 
action  cannot  be  produced  except  on  condition  that  there 
shall  be  a  stereochemical  relation  between  the  acting  sub- 
stance and  the  body  acted  upon. 

According  to  him,  it  is  necessary  that  the  ferments  and 
the  substances  they  act  upon  shall  have  a  like  geometrical 
structure,  or  at  least  a  certain  structural  resemblance. 

We  believe  that  this  hypothesis  will  explain  very  happily 
the  development  of  different  diastases  from  cells  which  have 
been  fed  with  different  substances. 

A  cell  nourished  by  starch  will  secrete  an  enzyme  having 
the  stereochemical  structure  of  starch,  while  if  the  cell  is 
nourished  by  cane-sugar,  the  diastase  which  it  will  form  will 
have  the  geometrical  constitution  of  cane-sugar. 

Our  knowledge  of  oxidizing  enzymes  is  much  less  exten- 
sive than  that  which  we  possess  of  hydrating  enzymes.  But 
the  facts  hitherto  observed  demonstrate  unquestionably  that 
in  this  case,  as  in  the  preceding,  there  are  found  to  be  present 
different  specific  ferments,  all  acting  like  oxidizing  agents, 
but,  individually,  on  different  materials.  For  this  class  of 
bodies  also,  it  is  evident  that  the  arrangement  of  the  various 
chemical  groups  in  the  molecules  of  the  oxidizable  substances 
considerably  influences  the  activity  of  the  enzymes.  Ex- 
amples are  known  of  oxidizing  enzymes  acting  on  an  entire 
series  of  homologous  bodies,  whose  action  is  still  possible,  if 
one  group  is  substituted  for  another,  while  the  action  of  these 


46  THE  ENZYMES   AND    THEIR  APPLICATIONS. 

same  enzymes  ceases  when  the  arrangement  of  the  groups  is 
changed.  Thus  it  is  that  laccase,  which  oxidizes  diphenol, 
its  homologues  and  the  products  of  substitution  of  these  sub- 
stances, exercises  its  action  on  all  their  derivatives  in  which 
the  two  hydroxyl  groups  are  found  in  the  ortho  position, 
while  the  same  diastase  does  not  act  on  the  isomeric  products 
in  which  these  groups  occupy  the  meta  position. 

Classification  of  Enzymes. — Now  that  we  have  acquired 
some  general  knowledge  of  enzymes  and  their  mode  of  ac- 
tion, we  can  turn  our  attention  to  the  individual  properties  of 
each  known  enzyme.  But  before  attempting  this  description, 
it  is  necessary  to  agree  upon  a  nomenclature  and  a  classifica- 
tion of  diastases.  The  chemists  who  discovered  the  first  dia- 
stases designated  them  by  very  different  names,  according  to 
their  different  points  of  view.  As  long  as  the  study  of  enzymes 
was  confined  to  a  small  number  of  substances,  the  drawbacks 
of  this  nomenclature  were  not  very  great.  But  at  the  pres- 
ent time  a  considerable  number  of  diastases  are  known,  and 
this  number  is  certain  to  increase.  Under  these  conditions 
it  would  be  desirable  to  have  a  logical  nomenclature  seeking 
to  designate  a  ferment  by  a  name  giving  a  clear  idea  of  its 
own  characteristics. 

Recognizing  this  need,  Duclaux  has  suggested  a  rational 
nomenclature,  by  designating  an  enzyme  by  the  name 
of  the  body  on  which  its  action  was  first  observed ;  and 
in  order  to  distinguish  the  substance  on  which  the  diastase 
performs  its  action  from  the  enzyme  itself,  he  has  proposed 
to  add  to  the  root  of  the  word  the  termination  asc.  Thus  the 
diastase  acting  on  casein  would  be  called  casease,  and  the 
diastase  which  transforms. starch  (amylum)  becomes  amylase. 

Unfortunately,  the  nomenclature  of  Duclaux  has  not 
been  adopted  by  all  workers,  and  a  certain  number  of  new 
diastases  have  received  from  their  discoverers  a  name  hav- 
ing, to  be  sure,  the  termination  asc,  but  whose  root  is  not 
derived  from  the  name  of  the  substance  on  which  the  diastase 
acts,  but  from  that  of  its  product.     Thus  the  glucase  of  Cuse- 


INDIVIDUALITY   OF   ENZYMES.  47 

nier  is  not  a  diastase  acting  on  glucose,  but  an  active  sub- 
stance transforming  starch  and  maltose  into  glucose.  This 
new  nomenclature  has  the  great  disadvantage  of  bringing 
about  confusion,  and  it  would  have  been  better  to  have  ad- 
hered to  the  nomenclature  of  Duclaux,  although  that  was  not 
all  that  could  be  desired.  It  is  not  well  to  take  as  the  root  of 
the  name  that  of  the  product  formed,  because  different  dia- 
stases may,  at  the  end  of  the  reaction,  produce  identical  re- 
sults, while  acting  on  very  different  bodies.  Thus  we  know, 
in  addition  to  glucase,  many  ferments  which  transform  cer- 
tain carbohydrates  into  glucose.  It  is  true  that  the  nomen- 
clature of  Duclaux  also  gives  rise  to  confusion.  Thus, 
the  action  of  glucase  was  first  observed  on  starch ;  one 
should  then  designate  this  enzyme  by  the  name  of  amylase,  a 
name  applied  to  the  diastase  from  malt.  It  is  then  necessary 
to  take  account,  not  only  of  the  substance  on  which  the  dia- 
stase acts,  but  also  of  the  substance  produced  by  the  diastase. 
From  this  point  of  view  one  would  name  the  glucase  of 
Cuisenier  amylo-glucase,  that  is  to  say,  indicating  that  it  is 
a  diastase  acting  on  starch  and  producing  glucose.  The 
diastase  of  malt,  on  the  contrary,  ought  to  be  called  amylo- 
maltase,  because  the  product  finally  resulting  from  the 
action  of  this  diastase  on  starch  is  maltose. 

However,  in  the  present  work  we  shall  retain  the  old 
nomenclature  and  give  to  the  diastases  the  names  which  are 
generally  met  in  the  literature.  The  reason  for  this  method 
is  that  we  know  very  well  that  every  change  of  nomenclature, 
although  aiming  to  simplify  matters,  only  adds  new  compli- 
cations, and  on  the  whole  produces  a  result  contrary  to  that 
which  was  intended.  The  most  rational  classification  of  en- 
zymes consists  in  distinguishing  them  according  to  the  chem- 
ical work  which  they  produce.  We  already  know  that  dia- 
stases can  produce  hydration,  oxidation,  or  molecular  trans- 
formation. We  shall  then  describe  the  diastases,  grouping 
them  according  to  the  chemical  character  of  their  action. 
The  study  of  the  diastases  of  proteid  matters  will  be  the  sub- 


48 


THE  ENZYMES  AND    THEIR  APPLICATIONS. 


ject  of  the  second  volume  of  the  present  work.  In  the  first 
we  shall  only  occupy  ourselves  with  the  diastases  producing 
either  hydration,  oxidation,  or  molecular  change. 

Hydrating  diastases  act  on  carbohydrates,  fatty  sub- 
stances, glucosides,  proteins,  and  urea. 

Oxidases  act  on  bodies  of  very  diverse  nature:  alcohols, 
phenols,  amides,  fatty  substances,  etc. 

Enzymes  causing  molecular  transformations  are  so  few 
that  not  many  bodies  susceptible  to  their  action  can  be 
named. 

CLASSIFICATION    OF    SOLUBLE    FERMENTS. 

A.  Soluble  Hydrating  Ferments. 
ist.   Soluble  Ferments  of  Carbohydrates. 


Names  of  the  Enzymes. 

Invertin  or  sucrase. 

Amylase  or  diastase. 

Glucase  or  maltase. 

Lactase. 

Trehalase. 

Inulase. 

Cytase. 

Pectase. 

Caroubinase. 


Substances  on  which  the 
Enzyme  Acts. 

Cane-sugar. 

Starch  and  dextrin. 

Dextrin  and  maltose. 

Lactose. 

Trehalose. 

Inulin. 

Cellulose. 

Pectin. 

Caroubin. 


Products  of  the  Reaction. 

Invert-sugar. 

Maltose. 

Dextrose. 

Dextrose  and  galactose. 

Glucose. 

Fructose,  levulose. 

Sugars. 

Pectates  and  sugars. 

Caroubinose. 


2d.    Soluble  Ferments  of  Glucosides. 


Emulsin. 

Myrosin. 
Betulase. 
Rhamnase. 


Steapsin. 
Lipase. 


Rennet. 

Plasmase. 

Casease. 

Pepsin. 

Trypsin. 

Papain. 


Amygdalin  and    other 
glucosides. 


Potassium  myronate. 

Gaultherin. 

Xanthorhamnin. 
jd.   Soluble  Ferments  of  Fatty  Substances 

>      Fatty  substances. 
4th.   Soluble  Ferments  of  Proteins. 


Glucose,  oil  of  bitter 
almonds,  and  hydro- 
cyanic acid. 

Glucose  and  allyl  iso- 
sulphocyanate. 

Oil  of  wintergreen. 

Glucose. 

Rhamnetine,  isodulcite. 


Glycerin  and  fatty 
acids. 


Caseinogen. 

(Casein,  Hammarsten.) 

Fibrinogen. 

Casein. 

Albuminoid  substances. 


Casein. 
(Para  casein.) 
Fibrin. 

Proteoses,  peptones. 
(  Proteoses,  peptones* 
\      amides. 


INDIVIDUALITY  OF  ENZYMES. 


49 


Urease. 

Laccase. 

Oxidin. 

Malase. 

Olease. 

Tyrosinase. 

Oenoxidase. 


jth.   Ferments  of  Urea. 

Urea.  |     Ammonium  carbonate. 

B.  Soluble  Oxidizing  Ferments. 


Uruschic  acid. 
Tannin,  anilin,  etc. 
Coloring  matters  of 

cereals. 
Coloring  matters  of 

fruits. 
Olive  oil. 
Tyrosin. 
Coloring  matter  of 

wine. 


Oxyuruschic  acid. 
Products  of  oxidation. 


Products  of  oxidation. 


C.  Ferment  Causing  Molecular  Decomposition. 


Zymase  or  alcoholic 
diastase. 


Various  sugars. 


Alcohol  and  carbonic 
acid. 


BIBLIOGRAPHY. 

Em.    Bourquelot. — Sur   l'identite   de  la   diastase   chez  les   etres   vivants. 

Comptes  Rendus  des  seances  de  la  Soc.  de  Biologie,  1885,  p.  73. 
Duclaux. — Individuality   des    diverses    diastases.    Microbiologic,    1883,   p. 

141- 

Em.  Fischer. — Einfluss  der  Configuration  auf  die  Wirkung  des  Enzymes. 

Berichte  der  deutschen  chemischen  Gesellschaft,  1894,  p.  2071,  2985, 

1429,  3479. 
Ueber  die  Verbindungen  des  Zuckers  mit  den  Alkohol  und  Ketonen. 

Berichte  der  deut.  chemischen  Gesellschaft,  1895,  p.   1145,  1429. 


CHAPTER  V. 

SUCRASE. 

Extraction  of  sucrase  from  yeast. — Secretion  by  Aspergillus  niger. — Prep- 
aration of  sucrase  in  the  dry  state. — Influence  of  the  quantity  and  of 
time. — Influence  of  temperature. — Difference  between  the  properties 
of  sucrase  of  different  origin. — Effect  of  the  acidity  or  alkalinity  of  the 
medium. — Action  of  oxygen  and  of  light. — Action  of  chemical  sub- 
stances.— Mode  cf  secretion  of  sucrase  in  the  cells. — Measurement  of 
sucrase. — Method  of  Fernbach. — Method  of  Effrcnt. 

Sucrase  is  a  diastase  capable  of  transforming  cane-sugar 
into  invert-sugar.  Saccharose,  under  the  action  of  sucrase, 
is  decomposed  with  addition  of  a  molecule  of  water,  giving 
two  monosaccharids :  dextrose  and  levulose, 

CjsHsaOn  +  H20  =  CGH1206  +  C6H1206. 

Saccharose.  Dextrose.  Levulose. 

Sucrase  is  very  widely  distributed  in  nature.  For  ex- 
ample, we  find  its  existence  in  the  saliva,  in  the  gastric  juice, 
and  in  the  small  intestine. 

Cane-sugar,  retained  for  some  time  in  the  mouth,  is  trans- 
formed by  the  action  of  the  saliva  into  invert-sugar.  How- 
ever, this  transformation  is  not  due  to  the  action  of  a 
secretion  of  the  salivary  glands,  but  rather  to  the  sucrase 
evolved  by  the  numerous  bacteria  which  are  found  in  the 
saliva,  for  the  active  substance  which  occurs  in  the  mouth 
transforms  only  very  limited  quantities  of  cane-sugar. 

The  diastases  of  the  gastric  juice  are  endowed  with  much 
stronger  inverting  power.  However,  in  spite  of  this  energy, 
the  inversion  of  saccharose  does  not  proceed  actively  in  the 

50 


SUCRASE.  5 1 

stomach.  A  considerable  part  of  the  cane-sugar  absorbed 
reaches  the  circulation  without  having  undergone  the  action 
of  the  diastases,  and  it  is  only  in  the  small  intestine  that  the 
transioi  iiiation  becomes  complete.  In  the  blood  the  pres- 
ence of  active  substances  capable  of  transforming  saccharose 
has  not  been  observed. 

Sugar  injected  in  the  veins  or  in  the  cellular  tissue  of  an 
animal  is  eliminated  in  the  urine;  but  this  elimination  does 
not  occur  when  sugar  is  injected  in  the  portal  vein.  It  then 
traverses  the  liver  and  there  undergoes  a  strong  diastatic  ac- 
tion which  completely  transforms  it. 

Sucrase  is  also  of  very  common  occurrence  in  the  vege- 
table kingdom:  it  is  found  in  buds  and  flowers,  as  well  as  the 
leaves  of  a  very  great  number  of  plants.  Furthermore, 
numerous  moulds,  such  as  Aspergillus  niger,  Mucor  raccmosus, 
Penicillium  glaucum,  Penkillium  Duclauxi,  Aspergillus  oryzae, 
yeasts,  and  many  other  ferments,  also  effect  the  inversion 
of  saccharose. 

As  a  general  rule,  a  cell  nourished  by  sugar  must  neces- 
sarily contain  a  sucrase.  This  rule  has,  however,  been  op- 
posed by  Hansen,  who  has  called  attention  to  the  fact  that 
the  mould  called  Monilia  Candida,  while  nourished  by  sac- 
charose, does  not  secrete  sucrase.  This  assertion  has  been 
successfully  refuted  by  E.  Fischer,  who  in  a  more  thorough 
study  of  this  mould  discovered  that  it  really  contains  a  su- 
crase, but  that  the  enzyme  is  retained  in  the  cells,  and  is  diffi- 
cult to  isolate. 

In  the  literature  sucrase  is  designated  under  different 
names:  it  is  called  glucose  ferment,  cytozymase,  zymase,  and 
invertin.  This  enzyme  was  discovered  by  Dobereiner  and 
Mitscherlich.  These  investigators  first  found  that  beer- 
yeast  inverted  saccharose.  They  also  remarked  that  this  ac- 
tive substance  can  be  extracted  from  the  yeast  by  washing 
with  water.  Berthelot  first  succeeded  in  isolating  the  dia- 
9tase  in  the  solid  form  by  precipitating  from  yeast  extract  by 
alcohol. 


52  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

J 

Mode  of  Preparation. — Several  different  methods  of 
preparation  of  sucrase  may  be  employed.  The  enzyme 
can  easily  be  obtained  by  putting  some  beer-yeast  in 
water  with  the  addition  of  a  few  drops  of  chloroform ;  after 
a  time  the  active  substance  is  dissolved  in  the  water.  Then 
the  liquid  is  filtered  to  remove  the  suspended  yeast-cells. 
The  solution  thus  obtained  is  necessarily  far  from  being  com- 
posed solely  of  sucrase,  the  yeast  containing,  besides  the  su- 
crase, other  extractive  matters  which  enter  into  solution  at 
the  same  time.  Notwithstanding  this,  the  infusion  is  very 
active,  and  may  very  well  serve  for  the  study  of  sucrase. 

A  better  method  of  preparation  of  this  enzyme  consists 
in  the  extraction  of  a  culture  of  Aspergillus  niger  in  Raulin's 
solution.  Yet,  the  extraction  of  the  diastase  from  Aspergillus 
niger  demands,  in  order  to  give  sufficient  quantities  of  en- 
zyme, the  maintenance  of  certain  conditions  without  which 
the  results  would  not  be  satisfactory.  The  best  method  of 
procedure  has  been  suggested  by  Duclaux.  He  advises  al- 
lowing a  culture  of  Aspergillus  niger  to  develop  on  a  large  sur- 
face of  Raulin's  solution  for  about  four  days,  and  when  the 
moulds  formed  have  taken  on  a  green  or  light  brown  color, 
drawing  off  the  liquid  and  replacing  it  by  pure  water  or  water 
containing  sugar.  On  this  new  liquid  one  allows  the  mould 
to  grow  further  for  two  or  three  days  up  to  the  complete  ex- 
haustion of  the  nutritive  medium.  Then  the  enzymes 
secreted  by  the  plants  enter  into  solution  and  it  only  remains 
to  filter  the  liquid  to  free  it  from  the  fragments  of  mould 
which  may  be  found  in  suspension  there.  The  solution  of 
sucrase  prepared  in  this  way  is  very  active  and  contains  rela- 
tively few  impurities.  To  prevent  the  liquid  changing  dur- 
ing the  growth  of  the  plant,  one  may  add  a  few  drops  of  mus- 
tard oil  which,  acting  as  an  antiseptic,  preserves  the  medium 
from  the  invasion  of  bacteria  without  destroying  the  diastase. 
However,  it  is  better  to  cultivate  the  mould  in  sterilized 
liquid,  and  to  inoculate  this  liquid  with  a  pure  culture  of 
Aspergillus  niger.     When  the  plant  has  sufficiently  developed 


SUCRASE.  53 

on  the  Raulin's  solution,  this  is  replaced  by  sterilized  distilled 
water. 

To  obtain  sucrase  in  a  dry  state,  E.  Donath  recommends 
the  following  method :  extract  the  beer-yeast  for  some  time 
in  absolute  alcohol ;  then  decant  the  alcohol,  filter,  and  dry  by 
exposing  to  the  air.  In  this  way  a  brittle  mass  is  obtained 
which  is  pulverized  and  treated  with  distilled  water.  This 
infusion  is  filtered  to  remove  the  yeast-cells  which  are 
present.  However,  as  the  cells  easily  pass  through  the  filter, 
one  must  make  sure  by  a  microscopic  examination  that  they 
have  all  disappeared  from  the  liquid.  If  cells  are  still  present, 
the  solution  must  be  filtered  several  times  through  a  double 
filter.  When  the  liquid  is  free  from  cells,  ether  is  added  and 
it  is  shaken.  A  viscid  substance  appears  which  remains  in 
suspension  in  the  upper  part  of  the  liquid  and  which  must  be 
separated  from  the  rest  of  the  infusion.  This  substance  is 
then  treated  with  distilled  water  and  dropped  slowly  into 
absolute  alcohol,  where  a  pulverulent  precipitate  is  produced. 
This  precipitate,  separated  from  the  liquid,  is  washed  -with 
alcohol  and  dried  in  vacuo.  This  procedure  furnishes  a  white 
powder,  swelling  in  water  and  dissolving  in  it  with  great  diffi- 
culty. It  can  be  kept  for  a  long  time  and  possesses  great 
diastatic  power.  It  appears  beyond  doubt,  however,  that  a 
considerable  part  of  the  active  substance  must  be  coagulated 
by  the  treatments  with  alcohol  and  ether,  and  consequently 
rendered  inactive. 

The  rapidity  of  inversion  of  saccharose  by  sucrase  de- 
pends upon  the  quantity  of  active  substance  employed,  as 
well  as  upon  the  physical  and  chemical  conditions  of  the 
medium  in  which  the  transformation  takes  place.  The  study 
of  special  conditions  which  favor  or  retard  diastatic  action  is 
still  more  interesting  in  that  it  furnishes  valuable  data  from 
the  theoretical  as  well  as  from  the  practical  point  of  view. 

We  shall,  therefore,  accord  to  this  question  the  develop- 
ment which  it  merits.  We  shall  first  study  the  influence 
exerted  on  the  rate  of  inversion  by  the  quantity  of  sucrase, 


54  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

and  the  temperature  at  which  it  works.  We  shall  next  de- 
termine the  time-factor  in  the  inversion,  as  well  as  the  in- 
fluence of  the  acidity  or  alkalinity  of  the  medium.  Finally, 
we  shall  see  how  light,  oxygen,  and  certain  other  chemical 
substances  influence  the  rate  of  the  transformation. 

Influence  of  Quantity  and  of  Time. — When  sucrase  acts 
on  a  solution  of  saccharose,  the  results  obtained  are  very  dif- 
ferent according  to  the  quantity  of  active  substance  em- 
ployed. 

If  one  holds  to  definite  conditions  one  may  observe  an 
almost  constant  relation  between  the  quantity  of  sucrase  em- 
ployed and  the  quantity  of  invert-sugar.  This  proportion 
is,  up  to  a  certain  point,  independent  of  the  concentration  of 
the  sugar  solution  in  which  the  diastase  works.  If,  for  ex- 
ample, i  and  2  cubic  centimetres  of  sucrase  are  made  "to  act 
during  the  same  time  and  at  the  same  temperature  upon 
equal  quantities  of  saccharose,  it  is  found  that  with  2  cubic 
centimetres  of  sucrase  twice  as  much  invert-sugar  is  obtained 
as  with  1  cubic  centimetre. 

However,  this  proportionality  between  the  quantity  of 
acting  substance  and  the  quantity  of  product  formed  is  not 
always  constant.  Duclaux  has  observed  that  the  law  of  pro- 
portionality is  true  only  when  the  sucrase  is  used  in  very 
small  amounts,  and  if  the  inversion  is  arrested  at  an  early 
stage.  The  ratio  holds  good  until  10  to  20  per  cent  of  the 
sugar  is  inverted,  after  which  it  fails.  When  one  studies  the 
influence  of  time  on  the  action  of  sucrase,  the  same  rule  holds 
good. 

Sucrase  is  a  very  energetic  enzyme.  According  to 
Duclaux,  1  gram  of  active  substance  transforms  4000  times 
its  weight  of  sugar.  However,  while  very  energetic,  the  ac- 
tion of  this  diastase  is  relatively  slow.  One  hundred  cubic 
centimetres  of  10  per  cent  solution  of  saccharose  to  which  1 
cubic  centimetre  of  sucrase  was  added,  when  kept  at  500, 
yielded  the  following  results : 


SUCRASE.  55 

After  i   hour 20  gr.   Invert-sugar. 

"       2  hours 41     " 

3       "      60     " 

"       4       "      80     " 

This  experiment  illustrates  the  remarkable  slowness  with 
which  inversion  is  produced.  It  is  noteworthy  in  this  con- 
nection that  the  amount  of  invert-sugar  increases  propor- 
tionally to  the  duration  of  the  action.  After  two  hours,  one 
finds  about  twice  as  much  invert-sugar  as  after  one  hour,  and 
after  five  hours  almost  five  times  as  much.  But  from  this 
time  on  the  proportion  ceases  to  exist.  If  we  continue  in 
fact  to  follow  the  action  of  sucrase  in  the  preceding  experi- 
ment, we  obtain: 

After   10  hours 1.72  gr.   Invert-sugar. 

"      20       "     3.12    " 

If  the  transformation  had  continued  with  the  same  speed 
as  at  the  beginning  of  the  action,  we  should  have  had : 

After   10  hours 2.00  gr.  Invert-sugar. 

"      20       "     4.00    " 

The  retardation  which  we  observe  commences  when  about 
20  per  cent  of  sugar  has  been  transformed,  and  in  proportion, 
as  the  inversion  proceeds,  the  abatement  continues  to  be 
marked.  The  irregular  course  which  we  observe  in  the  action 
of  sucrase  has  been  the  subject  of  different  researches  and 
has  occupied  many  investigators.  It  has  given  rise  to  various 
hypotheses  which  we  will  examine  later.  It  is  sufficient  now 
to  note  the  fact  before  we  pass  to  the  action  of  temperature. 

Influence  of  Temperature. — Temperature  plays  a  very 
important  part  in  the  inversion  of  saccharose,  and  exerts  a 
considerable  effect  on  the  degree  of  activity  of  sucrase.  At 
o°  sucrase  exercises  only  a  very  feeble  action,  but  it  in- 
creases considerably  with  increase  in  temperature.     This  in- 


56  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

crease  is  gradual  between  50  and  300.     Above  this  tempera- 
ture, from  300  to  500,  diastatic  activity  increases  rapidly. 

By  allowing  sucrase  from  yeast  to  act  for  an  hour  upon  a 
20  per  cent  solution  of  sugar,  we  have  obtained  with  the  same 
quantity  of  sucrase  at  different  temperatures  the  following 
figures : 

Temperature,  Gr.  Invert-sugar 

Degrees  Centigrade.  formed  per  ioo  c*c.  solution. 

o 

O O 

5° 0.05 

IO° O.I  I 

15° 0.18 

20° O.35 

3°° • 0.40 

40° 1.65 

500 2.20 

6o° 2.10 

The  temperature  at  which  inversion  proceeds  with  the 
greatest  rapidity  is,  according  to  Kjeldahl,  52. 50;  beyond 
that  the  diastase  becomes  more  and  more  weakened. 

In  trying  to  determine  the  temperature  at  which  sucrase 
is  destroyed,  it  is  important  to  have  conditions  absolutely 
constant,  because  the  concentration  of  the  liquid  and  the  re- 
action, as  well  as  the  other  special  qualities  of  the  medium, 
have  a  considerable  influence  on  the  activity  of  the  diastase. 

Sucrase  from  yeast,  much  diluted,  can  be  maintained  for 
an  hour  at  520  without  losing  its  inverting  power;  on  the 
contrary,  the  more  concentrated  solutions  of  sucrase  weaken 
very  perceptibly  when  they  are  kept  at  that  temperature  even 
for  a  short  time.  When  yeast  is  placed  for  an  hour  in  water 
at  650,  its  diastase  is  completely  destroyed ;  while  at  the  same 
temperature  a  part  of  the  active  substance  remains  un- 
changed, when  a  very  dilute  solution  of  sucrase  is  used.  The 
reason  for  this  difference  of  resistance  is  that  other  bodies 
unfavorable  to  the  action  of  sucrase  are  found  with  it  in  the 


SUCRASE.  5  7 

extract,  and  the  retarding  action  of  these  substances  evidently 
diminishes  with  the  degree  of  dilution  of  the  solution. 

The  presence  of  sugar  in  the  liquid  containing  sucrase 
perceptibly  increases  the  power  of  the  enzyme  to  resist  heat. 

On  the  whole,  the  variations  observed  between  the  opti- 
mum temperature  and  the  destructive  temperature  are  quite 
considerable.  The  optimum  temperature  is  found,  accord- 
ing to  different  authors,  between  500  and  560,  and  the 
destructive  temperature  between  650  and  700.  But  the  ac- 
tivity of  the  sucrase  is  weakened  as  the  destructive  tempera- 
ture is  approached. 

Sucrases  of  Different  Origin. — Kjeldahl  has  observed  that 
sucrase  extracted  from  bottom  yeasts  possesses  an  optimum 
temperature  different  from  that  of  the  active  substances  of 
top  yeasts.  For  the  latter  he  has  found  that  the  optimum 
temperature  is  3.50  higher  than  that  of  bottom  yeasts.  It  is 
not  only  the  optimum  temperature  which  varies  with  the 
origin  of  the  sucrase :  most  of  the  properties  of  the  enzyme 
depend  upon  its  origin  as  well  as  upon  the  mode  of  prepara- 
tion. Thus,  sucrase  extracted  from  yeasts  can  be  filtered  by 
the  Chamberland  filter,  while  the  active  substance  of  Asper- 
gillus niger  is  completely  held  back  by  the  filter. 

In  beer-yeast  sucrase  is  found  in  an  uneombined  state  and 
can  easily  be  extracted  by  water;  in  Monilia  Candida,  on  the 
contrary,  the  enzyme  is  retained  in  the  cells,  where  it  is  found 
combined  with  other  substances  which  render  it  insoluble. 
Sucrases  obtained  from  different  yeasts  may  also  differ  by 
their  greater  or  less  sensitiveness  to  chemical  reagents. 
Fernbach  has  found,  for  example,  that  the  enzyme  of  the 
yeast  of  Tantonville  is  fifty  times  more  sensitive  than  sucrase 
extracted  from  other  kinds  which  had  been  given  him  to 
study. 

These  differences  of  properties,  found  in  sucrase,  are  not 
confined  to  it.  We  shall  meet  with  similar  facts  when  we 
study  pepsin  as  well  as  many  other  soluble  ferments.  These 
differences  may  be  explained  by  the  presence  of  various 
foreign  substances  having  the  property  of  lowering  the  opti- 


.58  THE  ENZYMES  AND   THEIR  APPLICATIONS. 

mum  temperature  and  the  destructive  temperature  of  chang- 
ing the  solubility  of  enzymes,  and  of  influencing  their  sensi- 
tiveness towards  physical  and  chemical  agents. 

This  explanation  leads  to  the  conclusion  that  the  enzyme 
lias  in  itself  constant  properties,  and  that  if  two  sucrases,  for 
example,  show  different  characteristics  and  act  in  different 
ways,  one  must  simply  seek  the  cause  in  the  conditions  of  the 
medium, — in  the  presence  of  substances  endowed  with  an  ac- 
celerating or  retarding  power. 

But  all  authors  do  not  concur  in  this  opinion.  The  differ- 
ence which  exists  between  the  properties  of  two  enzymes  of 
the  same  nature,  but  of  different  origin,  has  sometimes  been 
interpreted  quite  otherwise.  One  may  suppose,  for  example, 
that  the  medium  in  which  the  enzyme  is  secreted  influences 
:not  only  the  mode  of  action,  but  also  the  very  composition  of 
the  diastase. 

By  this  hypothesis,  the  difference  existing  between  the 
modes  of  action  of  various  enzymes  must  be  regarded  as  re- 
sulting from  a  series  of  changes  in  the  composition  or  in  the 
structure  of  the  diastase,  whereby  one  simply  is  dealing  with 
"various  modifications  of  the  same  enzyme. 

We  have  just  said  that  sucrase  of  top  yeasts  produces  its 
maximum  effect  at  a  higher  temperature  than  the  optimum 
temperature  of  low  yeasts.  This  difference  may  be  attributed 
to  a  phenomenon  of  adaptation  of  the  yeast  to  the  medium 
in  which  it  works,  adaptation  having  as  its  consequence  the 
formation  of  different  diastases  at  the  different  temperatures. 
This  adaptation  to  the  medium  is  manifested  still  more 
clearly  when  one  studies  the  action  of  gastric  juice.  The 
pepsin  of  warm-blooded  animals  does  not  act  at  o°,  and  its 
maximum  effect  is  produced  at  500,  while  the  gastric  juice  of 
cold-blooded  animals  produces  a  manifest  action  at  o°,  and 
lias  an  optimum  temperature  of  400. 

Many  facts  analogous  to  the  preceding  are  known  which 
support  the  hypothesis  of  the  adaptation  of  diastases  to  their 
■environment. 


SUCRASE.  59 

But  the  existence  of  different  varieties  of  the  same  en- 
zyme is  very  difficult  to  demonstrate  exactly,  because  we 
always  have  to  deal  with  mixtures  of  enzymes  and  more  or 
less  clearly  recognized  foreign  substances.  However,  we 
are  the  more  led  to  deny  the  existence  of  different  varieties 
of  the  same  enzyme,  because  the  variations  in  the  properties 
of  the  same  enzyme  are  generally  not  very  pronounced,  and 
are  susceptible  of  being  artifically  reproduced  by  starting  with 
a  definite  diastase,  and  simply  changing  the  conditions  of  the 
medium.  We  think  it  more  logical  to  assume,  until  the  con- 
trary is  proved,  that  the  variations  observed  in  enzymes  of 
different  origin  are  due  to  the  presence  of  foreign  substances. 

We  shall  have  occasion  to  return  to  this  question  re- 
peatedly in  studying  the  enzymes  individually. 

Part  Played  by  the  Acidity  or  Alkalinity  of  the 
Medium. — The  acidity  or  alkalinity  of  the  medium  has  con- 
siderable influence  on  the  sucrase.  Kjeldahl  has  demon- 
strated that  a  slight  acidity  is  favorable  to  its  action,  while 
large  amounts  of  acid  or  alkali  diminish  its  diastatic  power. 

In  a  very  comprehensive  work,  Fernbach  has  studied  a 
sucrase  derived  from  Aspergillus  niger  and  has  examined  with 
great  care  the  influence  of  the  medium.  His  study  has  given 
valuable  information  on  the  question  which  we  have  to  con- 
sider. Fernbach  found  that  the  solution  of  sucrase  extracted 
from  Aspergillus  niger  always  possesses  an  acid  reaction  due 
to  the  oxalic  acid  elaborated,  in  greater  or  less  quantity,  by 
the  mould.  While  this  acid  reaction  is  in  reality  very  weak, 
the  diastatic  solution  may  have  dilute  soda  added  in  con- 
siderable amount  before  it  will  turn  litmus  paper  blue.  The 
sucrase  still  shows  itself  very  sensitive  to  the  action  of  quanti- 
ties of  alkali  too  weak  to  be  revealed  by  litmus  paper  and 
other  indicators  of  alkalinity.  Fernbach  made  the  follow- 
ing experiment  to  show  this  sensitiveness  to  the  acids  and 
alkalies  of  the  medium  : 

Into  each  of  eight  test-tubes  he  poured  two  cubic  cen- 
timetres of  an  infusion  of  sucrase,  added  to  each  of  them  a 


60  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

different  quantity  of  a  solution  of  soda  (1:15000),  then 
brought  the  volume  of  liquid  in  each  tube  up  to  10  cubic  cen- 
timetres by  the  addition  of  sugar  solution.  At  the  end  of 
an  hour  of  action  at  56°,  he  determined  the  quantity  of  invert- 
sugar  formed  in  each  of  the  tubes  and  obtained  the  following 
results : 

Numbers    of  Quantity  of  soda  Invert-sugar 

the   tubes.  added.  formed. 

1 occ 35  mgr. 

2 0.5 31 

3 1 • 25 

4 1-5 l7 

5 2 12 

6 ,:  2.5 7 

7. •■••' 3 5 

8 3-4 3 

The  liquid  in  the  tubes  1,  2,  3,  4,  showed  an  acid  reaction ; 
after  tube  4,  to  tube  7,  the  solution  was  neutral;  it  gave  a 
slightly  alkaline  reaction  in  tubes  7  and  8. 

We  notice  in  this  table  that  the  invert-sugar  formed 
diminishes  in  proportion  as  the  quantity  of  soda  in  the  solu- 
tion increases.  When  soda  is  not  added  35  milligrams  of 
invert-sugar  are  obtained,  while  the  addition  of  only  1.5  cubic 
centimetres  of  soda  (1  115000),  an  amount  hardly  sufficient  to 
neutralize  the  solution,  reduces  the  quantity  of  invert-sugar 
to  17  milligrams.  This  diminution  of  about  50  per  cent  is. 
here  effected  by  the  action  of  a  quantity  of  soda  equivalent 
to  about  1  gram  per  hundred  litres. 

This  extreme  sensitiveness  of  the  diastase  to  the  alka- 
linity or  acidity  of  the  medium  suggests  one  of  the  causes  of 
the  non-proportionality  between  the  quantity  of  active  sub- 
stance employed  in  an  inversion  and  the  quantity  of  invert- 
sugar  which  results.  In  fact,  when  the  sucrase  is  neutral  and 
used  in  small  amount,  the  quantity  of  matter  transformed  is, 
as  we  have  seen,  proportional  to  the  quantity  of  active  sub- 


SUCRASE. 


61 


stance  allowed  to  act,  but  this  proportion  ceases  to  exist 
when  the  experiments  are  made  with  an  acid  or  slightly  alka- 
line solution  of  sucrase.  It  is  evident  that  by  using  these 
increasing  quantities  of  sucrase,  there  are  at  the  same  time 
introduced  increasing  quantities  of  acid  or  alkali,  which  in- 
fluence more  and  more  strongly  the  degree  of  the  inversion 
and  change  the  ratio. 

Fernbach  has  determined,  in  his  work,  the  optimum 
amount  of  different  acids  for  the  greatest  activity  of  the  dia- 
stase. To  this  end  he  first  neutralized,  as  exactly  as  possible, 
a  sucrase  solution,  and  then  acidified  it,  using  increasing 
quantities  of  different  acids.  He  obtained  the  results  shown 
in  the  following  table : 


Acids. 

Optimum  quantity 
(No.  of  grams  per  litre). 

Inhibiting  quantity 
(No.  of  grams  per  litre). 

O.025 
I 

O.066 
2 

5 
10 

0.2 

2 

O.I 

4 
10 

50 

Lactic            "     

It  is  seen  that  the  optimum  amount  depends  upon  the  na- 
ture of  the  acid  employed. 

The  activity  of  the  enzyme  increases  in  the  presence  of 
small  amounts  of  acid  up  to  the  moment  when  the  optimum 
amount  is  reached;  but  this  once  passed,  the  presence  of  the 
acid  becomes  destructive  to  the  diastatic  action,  which  de- 
creases perceptibly. 

The  amount  of  oxalic  acid  producing  the  greatest  effect 
on  the  inversion  does  not,  by  itself,  possess  inverting  power 
at  560 ;  but  the  other  acids,  by  their  own  action,  invert  a  cer- 
tain amount  of  sugar.  The  invert-sugar  formed  in  the  pres- 
ence of  acids  results  then  from  the  combined  actions  of  the 
acid  and  of  the  diastase.  From  this  it  results  that  by  em- 
ploying different  acids,  each  in  its  own  optimum  amount, 
there  will  of  necessity  be  obtained  different  quantities  of  in- 


6  2 


THE  ENZYMES  AND    THEIR  APPLICATIONS. 


vert-sugar  with  the  same  amount  of  sucrase.  This  differ- 
ence is  due  to  the  action  of  the  acid  alone  and  not  to  that  of 
the  sucrase,  for  the  latter  is  always  influenced  in  the  same  de- 
gree by  the  different  acids. 

Fernbach  made  a  series  of  comparative  experiments  for 
studying  the  combined  actions  of  acid  and  sucrase.  He 
performed,  on  different  sugar  solutions,  two  experiments,  A 
and  B,  for  each  acid.  In  experiment  A  he  used  the 
optimum  amount  of  acid  with  the  addition  of  a  certain 
amount  of  sucrase;  in  experiment  B,  he  allowed  the  acid 
to  act  alone.  By  afterwards  determining  the  amount  of  in- 
vert-sugar formed  in  each  experiment,  he  has  been  able  by 
subtraction  to  determine  the  amount  of  invert-sugar  which 
may  be  attributed  to  the  special  actio(n  of  the  diastase.  These 
experiments,  carried  out  with  different  acids,  have  given  him 
the  following  results : 


Quantities  of  Acid 
per  litre. 

Sugar  Inverted  by 
Acid  and  Diastase. 

Sugar  Inverted 
by  Acid. 

Difference 

or  Sugar  Inverted 

by  Diastase. 

Sulphuric  acid.o.osgr. 
Oxalic           "      0.066 
Tartaric        "      1 
Succinic        "      2 
Lactic            "      5 
Acetic            "    10 

31-3 

30 

40 

34-2 

4i-5 

37-9 

0.7 

O 

8.6 

3-7 
12.2 

7.2 

30.5 

30 

31.4 

30.5 

29-3 

30.7 

It  will  be  seen  that  the  figures  of  the  last  column,  which 
designate  the  results  of  the  diastatic  action,  properly  speak- 
ing, are  the  same  for  all  the  acids,  for  the  slight  differences 
observed  may  be  attributed  to  errors  in  measurement.  This 
experiment  demonstrates  then  the  fact  that  we  have  stated 
above,  namely,  that  the  diastase  is  always  influenced  in  the 
same  degree  by  the  different  acids.  However,  the  data  fur- 
nished by  Fernbach  upon  the  influence  of  the  medium  apply 
exclusively  to  sucrase  secreted  by  Aspergillus  nigcr  cultivated 
on  Raulin's  solution.  It  is  probable  that  the  same  mould 
cultivated  in  other  media  would  furnish  sucrase  solutions  not 
having  the  same  sensitiveness  to  reagents.     Furthermore, 


SUCRASE.  63 

the  determination  of  the  amounts  of  acid  checking  or  favor- 
ing diastatic  action,  has  always  been  made  by  him  at  a  tem- 
perature of  560 ;  it  is,  therefore,  presumable  that  the  figures 
which  he  has  calculated  are  only  correct  for  that  temperature. 

In  reality,  from  300  to  400,  the  quantities  of  acid  which 
correspond  to  the  maximum  of  result  are  entirely  different 
from  the  amounts  necessary  at  a  temperature  of  560.  At 
these  temperatures  the  amounts  of  acid  must  be  multiplied 
by  5  to  produce  the  same  result  as  at  650.  According  to 
O'Sullivan  and  Tompson,  the  maximum  acid  amount  de- 
pends also  upon  the  quantity  of  sucrase  used,  for  they  have 
found  that  with  increased  amounts  of  sucrase,  increasing 
amounts  of  acid  must  also  be  Used. 

On  the  whole,  the  influence  of  the  reaction  of  the  medium 
on  the  rate  of  inversion  is  not  a  simple  one. 

Sucrase  derived  from  yeasts  differs  from  that  obtained 
from  Aspergillus  niger  in  the  resistance  to  the  action  of  acids. 
The  solution  of  sucrase  which  is  obtained  by  extracting  yeast 
with  cold  water  is  generally  more  sensitive  to  varying  reac- 
tions of  the  medium  than  the  diastatic  solution  extracted 
from  Aspergillus  niger.  The  sensitiveness  of  the  sucrase  of 
yeasts  varies,  furthermore,  with  the  nature  of  the  yeast  used, 
and,  for  the  same  yeast,  with  the  nutrition  to  which  it  has 
been  subjected. 

Fernbach  has  determined  the  amount  of  acid  which  is 
most  favorable  to  the  action  of  the  sucrase  in  three  kinds  of 
yeasts  (see  the  table  on  page  64).  It  is  seen  by  an  inspection 
of  this  table  that  the  maximum  amount  of  acid  is  about  0.2 
cubic  centimetres  for  the  champagne  yeast,  about  0.5  cubic 
centimetres  for  Saccharomyccs  Pastorianus  and  the  yeast  of 
pale  ale,  while  sucrase  extracted  from  Aspergillus  niger  gives 
a  maximum  result  only  when  much  larger  quantities  of  acid 
are  present. 

The  considerable  influence  which  the  content  of  alkali  in 
the  medium  exercises  on  the  course  of  transformation  has 
caused  it  to  be  supposed  that  the  accelerating  action  of  the 


64 


THE  ENZYMES  AND    THEIR   APPLICATIONS. 


acid  arises  from  a  modification  in  the  nature  of  the  enzyme 
brought  about  by  the  action  of  this  chemical  agent. 


Quantity  of  Acetic 
Acid  per  litre. 

Champagne  Yeast. 

Saccharomyces 
Pastorianus. 

Yeast  of  Pale  Ale. 

O 

33.3 

29.7 

18.8 

0.02 

38-7 

3i-9 

19.8 

O.05 

63-9 

32-4 

22.3 

O.I 

74-3 

32.4 

25-5 

0.2 

0.5 

79-4 

78.4 

32-9 
33 

28.3 
29.4 

I 

7-5 

31-3 

28.9 

2 

71.9 

29.6 

27.6 

5 

10 

50.4 

But  this  transformation  in  the  nature  of  the  diastase  is 
very  difficult  to  prove,  and  in  any  case  appears  to  be  slight. 

The  quantity  of  alkali  clearly  checking  the  inversion  does 
not  really  cause  an  appreciable  change  in  the  active  sub- 
stance. The  diminution  of  activity  is  due  to  the  abnormal 
conditions  of  the  medium,  rendered  refractory  to  the  action 
of  the  enzymes  by  the  addition  of  alkali.  But  the  active  sub- 
stance evidently  remains  unchanged  because  it  is  only  neces- 
sary to  neutralize  the  liquid  again  to  have  the  diastatic  work 
resumed  with  all  its  initial  intensity.  It  is  only  by  increasing 
the  amount  of  alkali  to  very  large  proportions  that  the  dia- 
stase is  destroyed,  just  as  albuminoid  substances  are 
destroyed  with  the  same  amounts  of  the  same  agents. 

Action  of  Oxygen  and  of  Light. — Duclaux  was  the  first 
to  find  that  air  exercised  a  very  appreciable  action  on  sucrase. 
He  observed  that  a  solution  of  sucrase  in  ordinary  water 
changed  color  in  contact  with  air  and  became  inactive  as  a 
result  of  oxidation. 

This  oxidation  of  sucrase  is  influenced  to  a  very  great  de- 
gree by  the  presence  or  absence  of  light,  as  well  as  by  the 
acidity  or  alkalinity  of  the  medium. 

Sheltered  from  light  and  in  a  slightly  alkaline  medium,  de- 


SUCRASE.  &5 

composition  by  the  oxygen  of  air  is  produced  very  rapidly ; 
it  is  less  pronounced  in  a  neutral  medium,  and  is  manifested 
slowly  in  the  presence  of  an  acid.  By  exposing  a  solution  of 
sucrase  to  the  action  of  the  air  at  350,  50  per  cent  of  the  active 
substance  is  destroyed  in  about  48  hours ;  at  a  temperature  of 
500  oxidation  is  more  rapid  and  the  same  degree  of  change 
is  reached  after  4  or  5  hours  exposure  to  oxygen.  Light 
alone,  in  the  absence  of  oxygen,  is  without  action  on  sucrase. 
Fernbach  has  shown  this  by  exposing  to  sunlight  exhausted 
tubes  containing  sucrase.  The  sucrase  remained  unchanged 
for  several  months. 

We  have  just  seen  that  in  darkness  acids  give  to  the  dia- 
stase a  considerable  resistance  to  the  action  of  air.  When 
sucrase  is  not  protected  from  light,  this  ceases  to  be  the  case 
and  there  are  alkalies  which  become  capable  of  protecting 
the  enzyme  against  oxidation.  By  leaving  in  contact  with  air 
and  light  two  solutions  of  sucrase,  one  slightly  acid  and  the 
other  slightly  alkaline,  it  is  found  that  the  acid  liquid  under- 
goes a  very  rapid  alteration,  while  the  alkaline  solution  is  pre- 
served for  a  long  time.  This  fact  has  been  observed  by 
Fernbach,  who,  by  exposing  three  sugar  solutions  of  different 
reactions  to  the  action  of  the  air  and  sunlight  for  48  hours, 
found  that  they  possessed  at  the  end  of  that  time  the  follow- 
ing diastatic  powers : 

Slightly  acid  solution 3.7 

Neutral  "        6.6 

Slightly  alkaline    "        7.4 

The  favorable  or  unfavorable  influence  of  the  acidity  or 
alkalinity  of  the  medium  on  the  oxidation  of  the  sucrase  has 
been  very  well  shown  by  the  following  experiment: 

Five  solutions  of  sucrase  showing  a  diastatic  power  of  18, 
some  acid,  others  alkaline,  in  different  degrees,  were  sub- 
jected, in  darkness,  to  the  action  of  the  air  at  a  temperature 
of  350  for  48  hours.  Determination  of  diastatic  powers  at 
the  end  of  the  experiment  gave  the  following  results : 


66  THE  ENZYMES  AND    THEIR  APPLICATIONS. 


Numbers 

of 

the 

Quantity  of  acid 

Diastatic 

experiments. 

in  millionths. 

power. 

I 

420 

18 

2 

270 

18 

3 

Neutral 

17 

4 

75  soda 

I4.6 

5 

150  soda 

IO.6 

We  clearly  see  from  this  table  the  preservative  action 
which  acids  exercise  and  also  the  destructive  influence  of 
alkalies. 

The  study  of  the  effects  of  light  and  oxygen  on  inversion 
of  saccharose  leads  us  to  a  practical  conclusion  relative  to  the 
preservation  of  the  diastase. 

To  preserve  a  solution  of  sucrase  it  is  of  foremost  import- 
ance to  avoid  oxidation  and  consequently  contact  with  air. 
For  this  purpose  a  vacuum  is  made  in  the  partially  filled  flask, 
or  else  the  diastatic  solution  is  covered  with  a  layer  of  oil.  I 
have  found  in  my  experiments  that  a  solution  of  sucrase  pre- 
pared in  this  way  still  possesses  all  its  energy  after  three 
months  of  preservation. 

Action  of  Chemical  Substances. — Sucrase  is  very  sensi- 
tive to  different  chemical  reagents.  Duclaux  has  completely 
elucidated  this  question  and  has  given  figures  which,  without 
being  absolute,  are  sufficiently  accurate. 

Calcium  chloride  markedly  suppresses  the  action  of  su- 
crase and  its  retarding  influence  increases  with  the  amount. 

Chlorides  of  sodium  and  potassium,  after  having  pro- 
duced a  favorable  effect  in  slight  quantities,  such  as  0.4  per 
cent,  lessen  the  diastatic  action  when  the  quantity  is  in- 
creased. 

Ammonium  chloride,  according  to  Nasse,  acts  very  favor- 
ably in  10  per  cent  solution,  and  in  small  amounts  is  indif- 
ferent. 

The  action  of  alkaline  salts  and  of  bases  is,  according  to 
Duclaux,  retarding  and  destructive  in  the  following  amounts : 


SUCROSE. 


67 


Salts. 

0.1% 

0.2% 

0.4* 

0.5* 

o.8£ 

4 
i-4 

7.2 

9-3 

25 
1 

5-6 

1-3 

Sodium  salicylate  up  to  0.2  per  cent  seems  to  be  without 
effect ;  with  0.4  per  cent,  the  diastatic  action  is  reduced,  for 
we  have  to  employ  1.3  of  the  diastase  instead  of  1.0  to  obtain 
the  same  effect.  Sodium  borate  and  sodium  arseniate  retard 
hydration  in  a  marked  degree.  The  presence  of  o.  1  per  cent 
reduces  the  efficiency  of  diastase  to  one-fourth  the  normal. 
Antiseptics  affect  very  differently  the  diastatic  power  of 
sucrase. 

Chloroform,  ether,  and  oil  of  wintergreen,  when  in  excess, 
reduce  the  activity  of  sucrase  by  about  ten  per  cent.  Toxic 
substances  have  also  an  inhibiting  action,  as  appears  from  the 
following  table  (Duclaux): 


Salts. 

Per  cent. 

o.oiif 

0.02# 

0.04^ 

O.I# 

0.256 

I.03 

I.30 

I6.3O 

I.O4 
1.25 

44 

1.25 
O.70 
62 

1.40 

Potassium  cyanide.. 

1.26 

Mercuric  chloride  has  then  a  very  slight  retarding  influ- 
ence; in  the  presence  of  0.1  per  cent,  the  diastase  is  but  lit- 
tle weakened. 

Potassium  cyanide  is  a  very  strong  retarding  agent ;  in 
the  presence  of  0.02  per  cent,  the  power  of  the  ferment  is  re- 
duced to  one-sixteenth  the  original.  Silver  nitrate  first 
checks,  then  accelerates  the  transformation,  due,  according 
to  Duclaux,  to  the  acidity  which  it  produces  in  the  sugar 
solution. 

Finally,  10  per  cent  alcohol  produces  a  retardation  ex- 
pressed by  the  figure  1.3.  Oil  of  garlic  and  other  essential  oils 
produce  only  inappreciable  effects  on  the  degree  of  inversion. 


68  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

Formation  of  Sucrase  in  Living  Cells. — We  have  seen 
above  that  all  cells  which  are  nourished  with  cane-sugar 
secrete  sucrase.  We  will  now  discuss  the  most  favorable 
conditions  for  the  secretion  of  this  enzyme  by  living  cells. 

In  the  cells  of  beer-yeast  cultivated  in  a  nutrient  solution 
containing  saccharose,  sucrase  appears.  To  explain  this 
phenomenon,  it  must  be  stated  that  the  cells  found  in  the 
presence  of  non-assimilable  substances  produce  a  secretion 
capable  of  transforming  these  substances  into  assimilable 
materials.  But  on  studying  the  phenomenon  more  closely, 
it  is  found  that  the  secretion  of  sucrase  does  not  strictly  de- 
pend on  the  manner  of  nutrition  of  the  cell ;  that  it  seems 
rather  to  be  intimately  allied  to  the  nature  of  the  organism, 
and  that  it  is  produced  independently  of  the  real  needs  of 
the  cell.  If,  for  example,  in  the  nutrient  solution  cane-sugar 
is  replaced  by  directly  assimilable  carbohydrate  the  secretion 
of  sucrase  continues.  In  this  case,  however,  the  nutrition 
does  not  at  all  necessitate  the  presence  of  this  enzyme. 
Though  the  nature  of  the  sugar  does  not  possess  any  influ- 
ence on  the  secretion  of  sucrase,  one  must  not  conclude  that, 
as  a  general  rule,  the  secretion  of  diastase  is  independent  of 
the  mode  of  nutrition  of  the  cell.  Experiment  has  shown,  on 
the  contrary,  that  the  diastatic  secretion  is  directly  allied 
to  the  nature  of  the  food,  while  being  independent  of  the 
carbohydrate  employed.  Yeasts  cultivated  in  beer-wort 
secrete  much  more  sucrase  than  yeasts  cultivated  simply  in 
sugar  solutions;  the  secretion  of  sucrase  is  favored  in  this 
case  by  the  nitrogenous  materials  of  the  malt.  Experiment 
has  shown,  for  example,  that  the  addition  of  peptones  in- 
creases the  quantity  of  sucrase  in  the  culture  medium. 

The  substances  most  favorable  for  the  growth  of  yeast  are 
not  always  those  which  most  favor  the  formation  of  sucrase 
Phosphates,  for  example,  which  influence  yeast  very  favor- 
ably are,  on  the  contrary,  unfavorable  to  the  formation  of  su- 
crase. Nitrogenous  materials  are  not  then  the  only  ones 
having  an  influence  on  the  secretion  of  sucrase.     Unfortu- 


SUCRASE. 


69 


nately,  the  conditions  which  favor  the  formation  of  diastase 
are  imperfectly  known.  They  merit  thorough  study,  for 
they  are  of  a  nature  to  afford  very  interesting  information 
from  a  theoretical  point  of  view. 

If  the  conditions  favorable  to  the  formation  of  sucrase  are 
little  known,  we  are  much  better  informed  as  to  the  manner 
of  diffusion  of  sucrase  through  the  cells. 

To  study  the  mode  of  formation  of  sucrase  in  Aspergillus 
niger,  Fernbach  proceeded  in  the  following  manner:  He 
sowed  a  certain  number  of  dishes  containing  equal  parts  of 
Raulin's  solution  with  a  definite  number  of  spores,  all  coming 
from  the  same  culture  of  Aspergillus.  He  then  subjected  the 
liquid  thus  sown  to  a  constant  temperature  of  350. 

He  determined  daily  in  one  of  his  experiments  the  weight 
of  the  plants,  the  sugar  remaining,  and  the  acidity,  as  well  as 
the  amount  of  sucrase  produced. 

Each  dish  contained  400  cubic  centimetres  of  Raulin's 
solution,  17.6  gr.  of  saccharose,  and  0.72  gr.  of  free  tartaric 
acid. 

The  results  which  he  obtained  are  tabulated  below : 


Weight 
of  the 
plant. 

Ash. 





3.105 

O.  116 

6.200 

O.171 

7-835 

O.191 

6.870 

0.200 

5-58o 

O.198 

By  following  in  this  table  the  figures  placed  under  the 
heading  "  Sucrase,"  it  is  seen  that  at  the  beginning  of  the 
development  of  the  young  plant,  when  it  is  using  great  quan- 
tities of  sugar,  the  sucrase  does  not  appear  in  the  culture 
liquid,  and  that  one  cannot  detect  its  appearance  until  the 
sugar  is  exhausted  and  inversion  no  longer  takes  place. 


7o 


THE  ENZYMES  AND    THEIR   APPLICATIONS. 


This  fact  is  of  great  interest ;  it  shows  us  that  inversion  is 
not  produced  in  the  liquid  which  surrounds  the  mould.  The 
presence  in  the  liquid  of  8.3  grams  of  invert-sugar  after  two 
days  tends  to  confirm  the  opinion  that  the  transformation 
takes  place  inside  the  cell.  If  we  accept  this  hypothesis,  we 
must  at  the  same  time  assume  that  sucrase  exists  in  the  cell 
from  the  beginning,  and  the  diffusion  noted  is  produced  as 
the  result  of  a  modification  of  the  cell  contents. 

In  fact,  Fernbach,  in  looking  for  sucrase  in  the  plant,, 
found  that  the  greatest  quantity  of  diastase  secreted  by  the 
cells  appeared  at  the  beginning  of  its  development,  and  that 
the  moment  of  its  appearance  outside  coincided  with  the  in- 
stant when  the  plant  had  already  caused  the  greatest  quantity 
of  sugar  to  disappear. 


After  1   day. .  . . 
"      2  days. . . 

"  3  "  •■• 
"  4  "  ... 
"      5      "    ... 


Saccha- 
rose re- 
maining-. 

Invert- 
sugar. 

Sugar 
con- 
sumed. 

Acidity. 

Sucrase 
of  the 
iiquid. 

Sucrase 
of  the 
cells. 

Weight 
of  the 
plant. 

I.36 

2.36 

O.92 

O.293 

2 

53 

O.65 

0.22 

I.65 

2-57 

O.368 

3 

47 

I.265 

O 

0.7 

3-74 

O.267 

5 

45 

I.78 

O 

O 

4.44 

O.143 

IO 

44 

I.65 

O 

O 

O.I35 

13 

35 

I. 6l 

The  diffusion  of  the  sucrase  at  the  moment  of  the  disap- 
pearance of  the  sugar  may  then  be  considered  as  a  conse- 
quence of  the  disintegration  of  the  plant.  When  we  reflect 
a  little,  we  realize  that,  in  cells  well  nourished  and  fortified 
with  reserve  food,  the  diffusion  must  be  accomplished  with 
great  difficulty. 

With  the  disappearance  of  the  invert-sugar  in  the  liquid, 
the  cells  begin  to  consume  their  reserve  food ;  vacuoles  are 
formed  and  filled  with  water,  which  certainly  facilitates 
diffusion. 

Moreover,  there  are  some  very  conclusive  experiments 
which  prove  that  the  diffusion  of  diastases  characterizes  a 
pathological  state  of  the  cells: 


SUCRASE.  7 1 

Place  two  young  and  identical  cultures  of  Aspergillus 
niger,  one  in  water,  the  other  in  a  rich  nutritive  medium. 
After  48  hours  examine  the  two  liquids  and  it  will  be  found 
that  the  first  medium  contains  a  great  quantity  of  active 
enzymes,  while  the  second  has  no  trace  of  any.  Denutrition 
then  favors  the  secretion  of  sucrase.  One  may,  moreover, 
take  a  culture  of  Aspergillus  niger  and  exclude  it  from  the  ac- 
tion of  the  air.  Thus  fructification  is  prevented  and  this  ab- 
normal condition  causes,  like  inanition,  an  abundant  diffu- 
sion of  the  diastase  in  the  culture  medium.  Finally,  beer- 
yeast  suspended  in  water  may  be  heated  for  several  seconds 
at  ioo°.  Thus  the  active  substance  is  completely  destroyed 
as  well  as  a  majority  of  the  yeast  cells.  On  allowing  the 
liquid  to  cool  one  notes,  after  a  while,  the  appearance  of  su- 
crase. The  secretion  of  the  enzyme  may  be  attributed  to  the 
cells  which  have  escaped  the  destructive  action  of  the  heat, 
although  greatly  injured  by  the  high  temperature  to  which 
they  have  been  subjected.  They  are  then  in  a  certain  path- 
ological state  and  diffuse  with  ease  the  active  substance  they 
contain. 

As  we  see  by  these  experiments,  the  lack  of  sugar  or 
oxygen,  the  elevation  of  the  temperature,  etc.,  may  equally 
favor  the  diffusion  of  the  sucrase  secreted  by  the  cells. 

Measurement  of  Sucrase. — We  may  easily  ascertain  the 
transformation  of  saccharose  into  invert-sugar  by  the  aid 
of  Fehling's  solution.  Cane-sugar  does  not  reduce  this  solu- 
tion, while  0.4941  gr.  of  invert-sugar  reduce  100  cubic  cen- 
timetres of  Fehling's  solution. 

The  transformation  of  saccharose  into  invert-sugar  may 
likewise  be  ascertained  by  the  change  of  optical  activity  that 
accompanies  the  transformation.  Cane-sugar  rotates  to  the 
right  and  the  mixture  produced  by  hydrolysis  on  the  con- 
trary, rotates  to  the  left.  Saccharose  gives  a  rotation  to  the 
right  of  aj-\-  73.8,  and  invert-sugar  a  rotation  to  the  left 
of  —  44.  The  measurement  of  sucrase  requires,  first,  the  de- 
termination of  the  quantity  of  invert-sugar.      Now,  the  same 


72  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

quantity  of  sucrase  may  furnish  greater  or  less  quantities  of 
invert-sugar.  These  variations  are  due  to  the  different  fac- 
tors which  we  have  indicated ;  the  reaction  of  the  medium, 
temperature,  duration  of  the  action,  etc.  It  must  not  be  for- 
gotten that  the  ratio  between  the  quantities  of  ferment 
employed  and  of  invert-sugar  obtained  only  exists  in 
early  stages  of  the  action,  and  before  20  per  cent  of  the  total 
amount  of  sugar  submitted  to  the  action  has  been  trans- 
formed. Hence,  it  is  absolutely  indispensable,  in  order  to  be 
able  to  compare  two  diastatic  products,  to  place  them  in 
identical  conditions.  To  avoid  errors  arising  from  the 
acidity,  care  must  be  taken  to  neutralize  the  liquid  as  exactly 
as  possible,  then  to  acidulate  with  1  per  cent  of  acetic  acid. 
The  choice  of  acetic  acid  is  not  arbitrary ;  it  is  due  to  the  fol- 
lowing reasons :  acetic  acid  can  be  used  in  considerable,  and, 
consequently,  easily  measureable  quantities.  It  does  not  dis- 
place the  other  organic  acids  of  the  solution,  and,  finally,  it 
has  little  influence  on  the  sucrase.  In  the  measurement,  the 
greatest  care  should  be  taken  to  prevent  oxidation  of  the 
sucrase,  and  to  this  end  the  analysis  should  be  made  as 
rapidly  as  possible.  Generally  one  should  let  the  sucrase  act 
only  for  one  hour. 

To  avoid  the  errors  which  may  come  from  the  deviations 
from  the  law  of  proportionality  between  the  quantity  of 
enzyme  used  and  the  quantity  of  sugar  inverted,  one  must 
seek  the  quantity  of  sucrase  capable  of  transforming  a  certain 
quantity  of  cane-sugar,  and  not  the  amount  of  sugar  which  a 
given  quantity  of  sucrase  can  invert. 

In  the  method  of  measurement  suggested  by  Fernbach, 
one  takes  as  the  unit  the  quantity  of  sucrase  capable  of  in- 
verting 20  centigrams  of  saccharose  in  one  hour  at  a  tem- 
perature of  560  in  the  presence  of  1  per  cent  of  acetic  acid. 
To  perform  this  measurement  the  solution  of  sucrase  is  pre- 
viously neutralized,  then  in  a  series  of  reaction  tubes,  each 
containing  4  cubic  centimetres  of  a  50  per  cent  solution  of 
saccharose,  is  added  1,  2,  3,  4,  5,  cubic  centimetres  of  the 


SUCRASE.  73 

sucrase  solution  to  be  analyzed ;  one  cubic  centimetre  of 
acetic  acid  (i  :io)  is  added  ;  the  volume  in  each  tube  is  brought 
up  to  10  cubic  centimetres.  The  tubes  are  left  for  one  hour 
at  a  temperature  of  560,  then  quickly  cooled;  and  several 
drops  of  a  soda  solution  are  added  to  arrest  inversion,  and 
the  quantity  of  invert-sugar  formed  in  each  is  estimated  by 
the  use  of  Fehling's  solution.  It  can  thus  be  seen  in  which 
of  the  tubes  the  20  centigrams  of  sugar  have  been  in- 
verted. 

Let  us  suppose  that  this  result  was  obtained  in  the  tube 
containing  5  cubic  centimetres  of  sucrase :  one  then  finds 
present  a  solution  containing  only  traces  of  saccharose. 

Since  the  amount  of  acetic  acid  used  in  the  experiment 
might  itself  have  inverted  some  centigrams  of  sugar,  it  may 
be  that  the  whole  of  the  inversion  is  due  to  foreign  substances 
and  not  to  the  diastase. 

In  order  to  be  certain  that  the  transformation  of  sac- 
charose is  due  to  the  effect  of  a  diastase,  the  experiment  must 
be  made  once  without  heating,  and  again  with  a  solution 
heated  to  ioo°,  to  see  if  the  results  are  the  same. 

In  case  1,  2  or  even  3  cubic  centimetres  of  solution  are 
sufficient  to  obtain  the  transformation  of  20  centigrams  of 
sugar,  the  inverting  power  of  the  solution  is  considerable, 
and  by  repeating  the  experiment  with  1^,  if,  2,  2.\,  etc.. 
cubic  centimetres  of  the  solution  experimented  with,  a  very 
accurate  measurement  of  the  diastatic  activity  may  be  ob- 
tained. 

In  case  \\  cubic  centimetres  of  solution  must  be  used  to 
obtain  20  centigrams  of  invert-sugar,  we  say  the  unit  amount 
of  sucrase  is  found  in  1^  cubic  centimetres  and,  consequently, 
that  the  solution  possesses  two-thirds  of  the  diastatic  power 
of  the  standard  solution. 

The  method  of  Fernbach  gives  fairly  accurate  results,  but 
it  demands  many  trials  and  a  long  series  of  measurements 
which  take  a  great  deal  of  time. 

When  it  is  a  question  of  a  qualitative  rather  than  quanti- 


74  THE  ENZYMES   AND    THEIR   APPLICATIONS. 

tative  estimate,  the  measurement  of  the  sugar  may  be  com- 
pletely omitted. 

In  order  to  test  for  sucrase  in  liquids  we  make  use  of  a 
very  expeditious  method  requiring  only  half  an  hour,  and  in 
which  the  inversion  is  ascertained  by  the  color  which  the  in- 
serted solution  gives  with  soda. 

For  this  kind  of  experiment  we  make  use  of  a  10  per  cent 
solution  of  sugar.  The  liquid  in  which  the  sucrase  is  meas- 
ured is  neutralized  as  accurately  as  possible  with  soda 
(i :  iooo).  In  two  test  tubes,  A  and  B,  are  poured  10  cubic 
centimetres  of  sugar  solution ;  to  A  is  added  one  cubic  cen- 
timetre of  diastatic  solution  and  to  B  one  cubic  centimetre  of 
the  same  solution  previously  heated  for  several  minutes  to 
ioo°.  The  two  tubes  are  left  for  thirty  minutes  at  500.  One 
cubic  centimetre  of  ordinary  soda  is  added  to  each  of  the 
lubes  and  heated  5  minutes  at  980.  If  a  solution  of  sucrase  is 
■present,  tube  A  takes  a  much  deeper  color  than  tube  B. 

It  is  possible  to  use  this  procedure  as  a  colorimetric 
method. 

BIBLIOGRAPHY. 

A.  Fernbach. — Recherches  sur  la  sucrase,  diastase  inversive  du  sucre  de 

canne.     These,  Paris,  1890. 
J.    Kjeldahl. — Recherches  sur  les  ferments   producteurs   de   sucre.    Mad- 

delelser  fra  Carlsberg  Laboratoriet,  Copenhague,  1879. 
Duclaux. — Microbiologic,  1883. 


CHAPTER  VI. 

SUCRASE    (Continued). 

Retarding  factors  and  their  explanation. — Deterioration  and  alteration  of 
sucrase. — Experiments  of  Effront  on  the  influence  exerted  by  invert- 
sugar  in  the  medium  in  which  the  inversion  is  produced. — 
Hypothesis  of  O'Sullivan  and  Tompson. — Arguments  for  and  against 
this  hypothesis. — Theory  of  Effront  on  the  decomposition  of  cane- 
sugar,  and  experiments  on  the  manner  of  action  of  acids  in  the  inver- 
sion of  saccharose. 

Factors    Retarding   Inversion    and    their    Explanation. 

— When  we  examined  the  course  of  the  transformation  of 
saccharose  by  sucrase,  we  noted  that  the  quantity  of  invert- 
sugar  formed  during  a  given  time  diminished  constantly  with 
the  progress  of  the  inversion.  This  diminution  is  produced 
in  such  a  manner  that  the  last  portions  of  saccharose  which 
remain  in  the  solution  are  transformed  very  slowly,  whereas 
at  the  beginning  of  the  transformation  the  inversion  is  ac- 
complished much  more  rapidly. 

Various  hypotheses  have  been  put  forth  for  explaining 
the  irregularities  which  are  observed  in  the  hydration  of 
cane-sugar.  Certain  authors  attribute  the  observed  retarda- 
tion to  a  deterioration  or  alteration  of  the  sucrase,  a  change 
taking  place  in  proportion  as  the  work  of  hydration  pro- 
gresses. According  to  other  authorities  the  retardation  in 
the  inversion  proceeds  from  the  disappearance  of  cane-sugar, 
whose  presence  favors  diastatic  action.  Finally,  the  hypoth- 
esis that  the  products  of  transformation  accumulated  in  the 
liquid  check  diastatic  action  may  give  a  probable  explanation 

75 


"2 6  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

of  the  irregularity  with  which  the  transformation  proceeds. 
In  this  last  hypothesis,  the  efficiency  of  the  enzyme  would  be 
hindered  by  the  invert-sugar  formed  during  the  action. 

Let  us  see  on  what  facts  these  different  hypotheses  rest, 
and  seek  a  rational  interpretation  of  the  irregular  course  of 
the  inversion. 

Deterioration  and  Alteration  of  the  Diastase.— The  hy- 
pothesis explaining  the  retarding  of  the  inversion  by  a  dete- 
rioration of  the  active  substance  during  its  work  does  not  ap- 
pear to  us  to  merit  a  serious  discussion.  The  ratio  observed 
at  the  beginning  between  the  duration  of  the  action  and  the 
quantity  of  sugar  transformed  affords  a  conclusive  proof  of 
the  permanence  of  the  diastase.  In  fact,  if  after  the  second 
hour  of  action  we  can  ascertain  that  the  quantity  of  sugar 
transformed  is  double  that  which  we  have  found  after  the 
first  hour,  it  is  very  evident  that  the  diastase  has  acted  during 
the  second  period  of  operation  with  the  same  energy  as  dur- 
ing the  first.  The  work  done  during  the  first  hour  has  not 
then  caused  any  destruction  of  the  active  substance,  and  it 
appears  difficult  to  us  to  believe  that  weakening,  which  is  not 
found  at  the  beginning,  can  be  produced  during  the  course  of 
the  operation. 

Moreover,  the  mode  of  action  of  enzymes  excludes  all 
idea  of  deterioration  of  the  active  substance  in  the  course  of 
transformation. 

In  studying  the  mode  of  action  of  amylase,  we  have  had 
occasion  to  make  evident  by  direct  experiments  the  enduring 
character  of  the  diastase  during  activity,  and  we  believe  that 
the  explanation  which  we  have  given  of  this  phenomenon  can 
be  generalized  and  extended  to  all  analogous  phenomena,  for 
the  slackening  has  identical  characteristics  in  a  great  number 
of  diastatic  actions. 

The  hypothesis  of  the  alteration  of  the  diastase  during  its 
activity  appears  to  be  very  probable.  In  fact,  numerous 
chemical  agents,  as  well  as  various  physical  conditions,  in- 
fluence the  sucrase  differently  and  to  a  very  great  degree. 


SUCRASE.  7  7 

In  the  experiments  -cited  in  the  preceding-  chapter,  for  ex- 
ample, the  slackening  in  the  transformation  must  unques- 
tionably be  attributed  to  the  combined  action  of  oxygen  and 
light. 

Still,  we  cannot  attribute  the  irregularity  in  inversion  en- 
tirely to  physical  or  chemical  causes,  for,  even  when  avoiding 
the  action  of  light  and  oxygen,  it  is  still  found  that  irregu- 
larity occurs. 

Furthermore,  the  alteration  of  sucrase  caused  by  oxygen 
does  not  become  appreciable  till  after  a  prolonged  contact 
with  the  air,  while  the  ratio  soon  ceases  to  hold  when  the 
diastatic  action  takes  place  in  a  very  dilute  solution  of  sugar, 
or  for  that  matter,  when  large  quantities  of  sucrase  are  put 
in  action. 

We  have  seen  that  in  placing,  under  certain  fixed  con- 
ditions, a  mass  of  sucrase  in  the  presence  of  any  quantity  of 
sugar  it  is  found  that  just  as  much  invert-sugar  is  formed 
during  the  second  hour  as  during  the  first. 

If,  under  the  same  conditions,  ten  times  as  much  sucrase 
is  employed,  this  equality  of  work  during  the  first  and  second 
hours  of  the  action  no  longer  holds  good,  but  the  proportion- 
ality will  be  observed  if  quantities  of  invert-sugar  are  com- 
pared after  10  and  20  minutes  of  work. 

If  the  amount  of  sucrase  is  again  augmented,  the  ratio  is 
found  to  hold  during  the  first  part  of  the  action,  but  to  cease 
after  ten  minutes. 

As  we  see,  the  retarding  force  may  appear  in  the  liquid  at 
different  times  according  to  the  amount  of  sucrase  used.  If 
then  we  accept  the  hypothesis  attributing  the  slackening  to 
an  alteration  of  the  active  substance,  we  must  assume  at  the 
same  time  that  the  same  sucrase  may  alter  very  rapidly  or 
very  slowly  according  as  it  is  used  in  a  large  or  small  amount. 

Finally,  as  the  proportionality  ceases  at  different  times 
for  the  same  quantity  of  sucrase  put  in  sugar  solutions  of 
different  densities,  it  must  be  assumed  that  the  quickness  of 
the  alteration  depends,  not  only  on  the  amount  of  enzyme 


78  THE  ENZYMES  AND    THEIR   APPLICATIONS. 

used,  but  also  on  the  concentration  of  the  sugar  solution. 
The  improbability  of  this  theory  can  be  seen. 

It  results  from  the  facts  which  we  have  just  presented  that 
neither  wearing  out  by  working  nor  alteration  by  physical 
or  chemical  agents  can  be  the  true  cause  of  the  slackening 
of  the  diastatic  action. 

Experiments  on  the  Influence  of  the  Invert-sugar. 
— Most  authors  have  attributed  the  failure  of  the  proportion- 
ality during  the  course  of  the  transformation  to  the  invert- 
sugar  formed,  which,  according  to  them,  checks  the  diastatic 
action. 

We  have  sought  to  verify  by  a  direct  experiment  this  re- 
tarding action  of  the  invert-sugar. 

To  this  end  two  solutions,  A  and  B,  were  made,  each  con- 
taining ioo  cubic  centimetres  of  water,  5  grams  of  sac- 
charose, 1  cubic  centimetre  of  acetic  acid,  and  10  cubic  cen- 
timetres of  yeast  sucrase.  In  solution  B  was  added  2  grams 
of  invert-sugar.  These  solutions  were  left  in  a  water-bath, 
and  from  time  to  time  samples  were  taken  in  which  the  quan- 
tity of  reducing  sugar  formed  was  determined. 

Solution  A.  Solution  B. 

Minutes.  Reducing  sugar  Reducing  sugar 

formed.  formed. 

15 O.26 O.25 

30 O.51 O.52 

45 0.79 0.74 

60 0.9    I.I  I 

90 1.2     1.2 

I20 I.4    I.32 

l8o -I-75 L89 

It  is  seen  from*  this  table  that  the  proportionality  ceased 
after  45  minutes  of  action  in  solution  A,  which  contained 
only  cane-sugar,  and  that  the  weakening  of  the  diastatic 
power  began  when  about  one-twentieth  of  the  total  sugar 


SUCRASE.  79 

contained  in  the  solution  had  been  transformed.  In  solution 
B,  which  contained  40  per  cent  of  invert-sugar  at  the  begin- 
ning" of  the  action,  inversion  was  not  at  all  retarded  during 
the  first  45  minutes.  On  the  contrary,  the  transformation 
appeared  to  conform  very  nearly  to  the  law  of  ratio,  and  the 
slackening  in  the  transformation  was  not  manifested  until 
after  an  hour  of  action. 

By  comparing  the  quantities  of  sugar  inverted  during 
the  first  hour  in  experiments  A  and  B,  we  find  that  only  18 
per  cent  of  saccharose  had  been  transformed  in  the  experi- 
ment made  with  pure  sugar,  and  22  per  cent  in  the  experi- 
ment made  with  a  mixture  of  saccharose  and  invert-sugar. 
The  slackening  in  the  action  of  the  diastases  must  not  then 
be  attributed  to  the  presence  of  the  products  of  transforma- 
tion in  the  medium  where  the  diastatic  work  is  performed. 

Hypothesis  of  O'Sullivan  and  Tompson. — O'Sullivan 
and  Tompson  have  put  forth  the  hypothesis  that  the  effect 
produced  by  sucrase  is  constantly  proportional  to  the  weight 
of  the  cane-sugar  present  in  the  liquid  at  the  time  of  action. 
Starting  from  that,  they  attribute  the  retardation  produced 
in  the  transformation  to  the  diminution  of  the  quantity  of 
saccharose  as  the  inversion  proceeds.  According  to  this 
view,  the  sucrase  would  act  in  the  same  way  and  with  the 
same  energy  from  the  beginning  to  the  end  of  the  action,  and 
the  slackening  would  be  exclusively  due  to  the  reduction  of 
the  supply  of  saccharose. 

Then,  if  we  invert  a  solution  containing  10  grams  of  sugar 
by  the  aid  of  a  quantity  of  sucrase  able  to  produce  a  gram  of 
invert-sugar  in  the  first  10  minutes,  we  may  expect  to  have 
produced  during  each  of  the  following  ten  minutes  a  hydra- 
tion corresponding  to  one-tenth  of  the  total  quantity  of  cane- 
sugar  contained  in  the  solution  at  that  time. 

According  to  this  theory,  the  mode  of  action  of  sucrase 
would  not  change  during  the  work;  the  course  of  the  inver- 
sion would  be  on  the  whole  regular  and  the  slackening  ob- 
served would  be  the  direct  and  inevitable  consequence  of  the 


So  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

regularity  of  the  phenomenon  itself.  For,  if  after  the  first 
10  minutes  of  the  action  we  have  found  the  production  of  one 
gram  of  invert-sugar,  after  the  following  10  minutes,  we 
shall  have  obtained  only  0.9  grams,  inasmuch  as  the  action  is 
produced  in  this  case  no  longer  upon  10  grams,  but  only 
upon  9  grams  of  cane-sugar.  After  20  minutes,  there  will 
remain  in  the  solution  8.1  grams  of  saccharose  and,  by  acting 
always  under  the  same  conditions,  the  sucrase  will  invert  dur- 
ing the  following  10  minutes  10  per  cent  of  the  sugar  remain- 
ing, or  0.81  grams. 

This  hypothesis  is,  these  authors  say,  fully  confirmed  by 
the  measurement  of  the  quantities  of  sugar  inverted  at  the 
end  of  periods  varying  in  arithmetical  progression. 

Arguments  for  and  Against  this  Hypothesis. — The 
theory  of  O'Sullivan  and  Tompson  is  very  attractive;  it  has. 
not,  however,  found  many  adherents,  and  various  objections 
have  been  raised  against  it. 

First,  it  has  been  objected  that  the  experimental  proofs 
which  they  bring  forward  in  favor  of  their  theory  do  not  at  all 
prove  that  the  slackening  comes  from  the  diminution  of  the 
quantity  of  cane-sugar.  In  fact,  the  results  of  their  experi- 
ments may  equally  well  be  explained  by  the  gradual  increase, 
during  the  action  of  the  sucrase,  of  the  quantity  oi  invert- 
sugar. 

Our  experiments,  cited  above,  on  the  influence  of  the  in- 
vert-sugar, show  the  invalidity  of  this  argument. 

But,  still  another  objection  may  be  raised  to  the  theory  of 
O'Sullivan  and  Tompson.  If  the  gradual  disappearance  of 
cane-sugar  is  really  the  retarding  cause,  the  quantity  of  sugar 
inverted  by  any  amount  of  sucrase  will  be  in  direct  relation 
with  the  weight  of  the  cane-sugar  present  in  the  liquid.  In- 
crease in  the  amount  of  cane-sugar  will  then  cause  a  corre- 
sponding increase  in  the  amount  of  sugar  inverted.  We  al- 
ready know  that  these  expectations  are  not  always  realized, 
and  that  the  same  quantity  of  sucrase  produces  the  same 
amount  of  invert-sugar,  independently  of  the  concentration 


SUCRASE.  St 

of  the  sugar  solution.  There,  then,  is  a  serious  argument 
against  the  hypothesis  that  we  are  considering,  but  it  is  none 
the  less  true  that  the  quantity  of  sugar  contained  in  the 
medium  is  not  without  influence  on  the  slackening.  By 
studying  the  phenomenon  more  closely,  we  find  that  the  de- 
gree of  hydration  depends  upon  two  factors. 

At  the  beginning  of  the  action  it  is  the  quantity  of  su- 
crase  used  which  plays  a  predominant  part,  and  the  quantity 
of  invert-sugar  formed  is  proportional  to  the  quantity  of  ac- 
tive substance  used.  When  the  inversion  is  more  advanced, 
the  influence  of  the  quantity  of  sucrase  becomes  less.  The 
transformation  becomes  directly  proportional  to  the  sugar 
content. 

The  successive  influence  of  the  two  factors  may  be  shown 
by  the  following  experiment: 

To  ioo  cubic  centimetres  of  three  liquids,  A,  B,  and  C, 
containing  respectively  5,  10,  and  20  grams  of  saccharose, 
add  the  same  quantity  of  sucrase.  Then  leave  these  solu- 
tions in  a  water-bath  at  a  temperature  of  500.  From  time  to 
time,  take  samples,  and  determine  the  saccharose  remaining 
in  them  and,  when  in  solution  A  15  per  cent  of  saccharose 
has  been  transformed,  begin  to  measure  the  invert-sugar  in 
the  two  other  specimens.  There  is  then  obtained  the  follow- 
ing figures : 

Invert-sugar  at  the  end  of  A  B  C 

2  hours 0.75         0.74         0.78 

4     "       1.1  1.4  1.6 

One  finds  then  at  the  beginning  of  the  action  almost  the 
same  quantities  of  sugar  transformed  in  the  three  liquids,  A, 
B,  and  C,  but  at  the  end  of  four  hours  the  conditions  change 
and  there  is  found  in  the  solution  containing  20  per  cent  of 
sugar  1.6  gr.  of  transformed  sugar,  while  the  5  per  cent  liquid 
affords  only  1.1  gr.  of  invert-sugar. 

Th«  concentration  of  the  sugar  solution,  then,  influences 


&2  THE  ENZYMES  AND    THEIR   APPLICATIONS. 

the  action  of  the  sucrase  up  to  a  certain  point.  The  course 
of  the  hydration  of  the  sugar  in  solutions  of  different  concen- 
trations is  shown  to  be  rather  favorable  to  the  theory  of 
O'Sullivan  and  Tompson,  especially  if  the  beginning  of  the 
transformation  is  excluded. 

Still  this  theory  does  not  appear  to  us  to  be  based  on 
very  well  established  data. 

By  noting  the  transformation  of  the  sugar  at  different 
periods  we  have,  indeed,  found  that  the  slackening  in  the  in- 
version increases  in  proportion  as  the  action  advances,  but 
we  have  never  been  able  to  see  the  regularity  which  the 
authors  of  the  hypothesis  claim,  and  which  is  the  very  basis 
of  their  theory. 

Even  admitting  that  it  can  be  demonstrated  experiment- 
ally that  the  decrease  in  the  inversion  varies  in  geometric 
progression,  this  demonstration  would  show  the  way  in 
which  the  retardation  takes  place,  but  it  would  not  at  all  re- 
veal the  real  cause.  By  making  the  same  quantity  of  sucrase 
act  on  sugar  solutions  of  different  concentration,  it  is  seen 
that  the  retarding  force  is  manifested  very  differently.  In  a 
dilute  solution,  the  proportionality  between  the  duration  of 
the  action  and  the. quantity  of  sugar  formed  is  lost  at  the  end 
of  a  relatively  short  time.  In  a  concentrated  solution,  on 
the  contrary,  it  persists  for  a  longer  time. 

These  great  differences  in  the  action  of  sucrase  are  easily 
explained  if  one  determines  in  liquids  of  different  concentra- 
tion the  quantitative  relation  which  exists  between  the  sugar 
inverted  and  that  which  is  not  yet  inverted. 

By  studying  the  variations  of  this  relation  for  a  dilute 
solution  and  a  concentrated  solution,  it  is  found  that  the 
slackening  of  hydration  does  not  become  really  appreciable 
until  the  sugar  solutions  contain  about  15  parts  of  invert- 
rugar  to  85  parts  of  unchanged  saccharose. 

It  being  granted  that  sucrase  produces,  at  the  beginning 

f  the  transformation  a  hydrating  effect  proportional  to  its 

iiuantity,  it  is  quite  evident  that  in  the  dilute  solution  the  ratio 


SUCRASE.  83 

-|4  will  be  reached  much  more  quickly  than  in  the  concen- 
trated solution. 

In  other  words,  when  the  slackening  of  hydration  is  first 
observed,  the  concentrated  solution  contains  more  invert- 
sug-ar  than  the  dilute  solution,  although  the  quantity  of  su- 
crase  put  in  action  would  be  the  same  in  the  two  solutions. 

The  retardation  in  the  inversion  depends  directly  upon  the 
composition  of  the  liquid  in  which  the  diastase  acts.  It 
is  not  caused  by  the  diminution  of  the  quantity  of  cane-sugar 
contained  in  the  solution,  and  neither  does  it  arise  from  the 
increase  of  the  quantity  of  invert-sugar.  It  is  rather  caused 
by  the  combination  of  the  two  circumstances. 

Theory  of  Inversion  of  Cane-sugar. — We  must,  we  be- 
lieve, seek  in  the  structure  of  the  molecules  of  saccharose  the 
real  origin  of  the  retarding  force.  It  is  generally  supposed 
that  the  action  of  the  sucrase  is  manifested  by  the  successive 
hydration  of  molecules  of  sugar  with  which  it  comes  in  con- 
tact. It  is,  however,  probable  that  the  mechanism  of  inver- 
sion does  not  possess  this  simple  character. 

It  is  more  probable  that  the  sucrase  acts  from  the  begin- 
ning on  the  whole  mass  of  sugar  with  which  it  comes  in  con- 
tact, and  that,  in  the  transformation  of  cane-sugar  into  in- 
vert-sugar, it  produces  a  series  of  modifications.  It  is  easily 
conceived  that,  by  successive  hydrations,  there  may  be 
formed,  besides  invert-sugar,  a  series  of  substances  very 
similar  to  saccharose,  but  which  may  have  a  different  degree 
of  sensitiveness  to  the  sucrase.  Such  are  the  intermediate 
substances  produced  by  hydration  which  afterwards  appear 
more  or  less  suitable  for  the  transformation,  and  it  is  from 
this  greater  or  less  susceptibility  to  hydration  that  the  check- 
ing of  the  inversion  comes. 

It  may  be,  too,  that  the  changes  undergone  by  the  sac- 
charose consist  in  changes  in  the  configuration  of  the  mole- 
cules, and  that  stereochemical  isomers  are  formed  in  the 
solution. 

Still  it  is  impossible  for  us  to  bring  forward  convincing 


84  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

facts  in  favor  of  our  hypothesis.  Having  predicted  the 
formation,  in  the  course  of  inversion,  of  products  inter- 
mediate between  the  saccharose  and  the  invert-sugar,  we 
have  sought  to  isolate  these  products,  or  at  least  to  charac- 
terize them.  But  the  various  experiments  attempted  with 
this  in  view  have  remained  without  results.  Still  the  hy- 
pothesis, as  we  shall  see,  finds  support  in  the  process  of  hy- 
dration by  acids. 

Experiments  on  Transformation  by  Acids. — By  studying 
the  inversion  of  sugar  in  the  presence  of  increasing  amounts 
of  acid,  we  have  been  able  to  ascertain  that  there  is  produced 
at  certain  periods  a  noticeable  retardation  in  the  progress  of 
hydration.  The  slackening  takes  place  at  times  which  always 
coincide  with  certain  stages  in  the  hydration  of  saccharose. 
Thus  there  is  a  striking  analogy  between  the  action  of  acids 
and  that  of  sucrase.  The  retarding  force  is  found  in  both 
cases,  in  the  action  of  the  acids  as  well  as  in  the  diastatic  ac- 
tion, and  the  moment  when  the  slackening  begins  corre- 
sponds to  the  instant  when  the  relation  between  the  quanti- 
ties of  invert-sugar  and  of  non-transformed  sugar  reaches  a 
certain  value. 

This  resemblance  between  the  mode  of  action  of  chemical 
and  of  physical  agents  proves  that  the  retarding  force  does 
not  come  from  the  sucrase,  and  that  the  origin  of  the  retarda- 
tion is  not  necessarily  to  be  attributed  to  the  manner  of  de- 
composition of  the  cane-sugar  and  to  the  formation  of  transi- 
tory products  which  resist  differently  the  agents  of  trans- 
formation. 

Some  details  of  the  experiments  which  we  have  made  are 
here  given : 

Dissolve  a  gram  of  cane-sugar  in  distilled  water,  add  2 

n 
cubic  centimetres  of    —  sulphuric  acid,  and  bring  the  volume 
10       F  & 

up  to  100  cubic  centimetres.  Leave  in  water-bath  at  6o°,  for 
one  hour,  then  neutralize  accurately  with  normal  soda,  and 
determine  the  quantity  of  invert-sugar  formed  by  the  action 


SUCRASE. 


85 


of  the  acid.     This  experiment  is  then  repeated  with  4,  6,  8, 

n 
10,  etc.,  cubic  centimetres  of  —  sulphuric  acid,  and  the  fol- 
io       L 

lowing;'  results  are  obtained : 


Cubic  centimetres 
of  acid. 


Percentage  of 
invert-sugar. 

.  ..     5.71... 

..  .II.36..  . 


6 15.29. 

8 22.12. 

10 26.34. 

12 32.00. 

14 37-!4- 

16 46.76. 

18 51.36. 

20 53.33. 

22 52.00 

44 


65.20 


Increase. 

••571 
••5.65 

••3-93 
..6.83 

.  .4.22 

..5.16 

..5.14 
.  .9.62 
.  .4.60 
..1.97 

••i-33 
.  .  1.20 


The  heading-  "  Percentage  of  invert-sugar  "  indicates  the 
quantity  of  sugar  transformed  during  the  experiment. 

Under  the  heading  "  Increase  "  we  have  written  the  in- 
crease in  the  quantity  of  sugar  transformed  by  each  addition 
of  two  cubic  centimetres  of  acid. 

By  following,  in  the  table,  the  progress  of  the  hydration 
in  the  presence  of  increasing  amounts  of  acid,  it  is  found 
that  the  ratio  between  the  quantities  of  acid  and  invert-sugar 
formed  is  not  at  all  constant.  This  ratio  exists  in  the  first 
experiments  and  disappears  completely  in  those  where  50 
per  cent  of  cane-sugar  is  transformed.  Thus  2  cubic  cen- 
timetres of  acid  have  formed  5.71  centigrams  of  invert-sugar; 
with  a  double  amount,  11.36  eg.  of  invert-sugar  are 
formed,  or  practically  double  the  preceding  quantity.  Tf  we 
increase  the  amount  of  acid  and  use  20  cubic  centimetres  we 
cause  a  hydration  of  53  per  cent  of  sugar;  but  beyond  this 
amount  of  acid  the  hydration  slackens  and  44  cubic  cen- 


S6  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

timetres  of  acid  hydrate  only  65  per  cent  of  the  cane-sugar 
present  in  the  liquid.  If  the  ratio  really  existed,  with  this 
amount  of  acid  a  complete  inversion  of  all  the  saccharose 
contained  in  the  liquid  would  be  obtained. 

The  action  of  increasing  amounts  of  acid  is  still  better 
shown  when  the.  heading  "Increase"  is  followed.  In  the 
first  experiments  the  increase  gradually  falls  from  5.71  to 
3.93,  but  in  the  following  ones,  when  nearly  a  quarter  of  the 
whole  quantity  of  cane-sugar  has  been  transformed,  a  com- 
plete change  is  found  in  the  progress  of  the  inversion.  The 
increase  rises  to  6.83  to  fall  again  to  5.14. 

The  increase  is  again  augmented  when  half  the  cane-sugar 
has  been  transformed,  then  it  undergoes  another  decline,  and 
is  represented  by  the  number  1.2  in  the  presence  of  65  per 
cent  of  invert-sugar. 

The  course  of  hydration  by  acids  is  then  far  from  being 
regular.  A  great  number  of  analogous  experiments,  made 
under  the  same  conditions,  have  always  confirmed  in  our 
work  the  non-existence  of  a  ratio  between  the  quantities  of 
acid  used  and  invert-sugar  formed.  We  have  always  found  a 
slackening  in  hydration,  which  coincides  with  the  appearance 
of  a  definite  ratio  between  the  quantities  of  sugar  inverted 
and  of  non-transformed  sugar  in  the  liquid. 

The  action  of  acids  is  then,  in  the  main,  identical  with  that 
of  the  diastase,  and  the  retardation  observed  in  hydration  by 
sucrase  must  rather  be  attributed  to  a  structural  transforma- 
tion of  the  saccharose  molecule. 

BIBLIOGRAPHY. 

Mitscherlich. — Rapport  annuel  de  Berzelius.     Paris,  1843. 

Berthelot. — Sur  la  fermentation  glucosique  du  sucre  de   canne.     Chim. 

org.  fondee  sur  la  synthese.     Paris. 

Comptes  Rendus,  L.  i860,  p.  980. 

Dumas.— Sur  les  ferments  appartenant  au  groupe  de  la  diastase.  Comptes 

Rendus.  1872. 
Nasse.— Bemerkungen  zur  Physiologie  der  Kohlenhydrate.     Pfliig.  Arch., 

1877. 


SUCRASE.  8  j 

J.  O'Sullivan. — L'invertase  de  la  levure  de  biere,  1893.     Monit.  scient. 

J.  Kjeldahl. — Carlsberger  Laboratorium,  1879  et  1881. 

E.  Donath. — Ueber  den  invertirenden  Bestandtheile  der  Hefe.     Berichte 

der  deutsch.  chemisch.  Gesellschaft,  1875,  VIII,  p.  975. 
Kossman. — Etudes   sur  les  ferments  solubles   contenus  dans  les  plantes. 

Comptes  Rendus,  2e  ser.,  1875,  p.  406. 
Em.  Bourquelot. — Sur  la  physiologie  du  gentianose  et  son  dedoublement 

par  las  ferments  solubles.     Comptes  Rendus,   1898,  p.   1045. 
O'Sullivan     et    Tompson. — Sur     un     ferment     non-organise,    l'invertase. 

Comptes  Rendus,   1872,  2e  ser.,  p.  295. 
Fernbach. — Recherches    sur   la   sucrase,    diastase    inversive   du    sucre    de 

canne.    These,  Paris,  1890. 
Duclaux. — Sur  Taction  de  la  diastase.     Annales  de  l'lnstitut  Pasteur,  1897. 
J.  O'Sullivan. — L'invertase.  Journal  of  the  Chem.  Soc,  1890,  I,  p.  834-931. 
Beitriige    zur    Geschichte    eines    Enzymes.     Berichte    der    deutsch. 

chemisch.  Gesellschaft,  1890,  p.  743. 
Ad.  Mayer. — Die  Lehre  von  den  chemischen  Fermenten  oder  Enzymol- 

ogie.     Heidelberg,  1882. 
Wasserzug. — Ann.  de  l'lnstitut  Pasteur,  1887. 
E.  Fischer  et  Lintner. — Verhalten  der  Enzyme  gegen  Melibiose,   Rohr- 

zucker  und  Maltose.     Berichte  der  deutsch.  chemisch.  Gesellschaft, 

1895,  3.  3055- 
Miura. — Inversion    des    Rohrzuckers.     Berichte    der    deutsch.    chemisch. 

Gesellschaft,  1895,  p.  623. 
V.  Tieghem. — Inversion  du  sucre  de  canne  par  le  pollen.     Societe  bota- 

nique  de  France,  1866. 


CHAPTER  VII. 

FERMENTATION  OF  MOLASSES. 
Industrial  application  of  sucrase. — Fermentation  of  molasses. 

The  enzyme  producing  the  inversion  of  saccharose  does 
not  constitute  a  commercial  article.  It  has  a  very  limited 
manufacture  and  serves  exclusively  for  study  and  for  labora- 
tory experiments.  But.  if  specially  prepared  sucrase  is  not 
used  in  industrial  operations,  this  diastase  nevertheless  plays 
an  important  part  in  fermentations,  and  especially  in  the 
manufacture  of  alcohol  from  molasses. 

The  fermentation  of  molasses,  a  substance  which  contains 
nearly  50  per  cent  of  saccharose,  is  a  relatively  simple  opera- 
tion.    It  is  effected  in  the  following  manner : 

The  molasses  is  first  diluted  with  water  acidified  with  sul- 
phuric acid,  and  brought  to  from  90  to  12°  Beaume. 

Thus  a  mash  is  formed  ready  to  undergo  the  action  of  the 
beer-yeast  which  inverts  the  saccharose  and  causes  the  in- 
vert-sugar produced  to  ferment.  The  transformation  of 
molasses  into  alcohol  appears  then  to  be  a  simple  industrial 
operation.  The  apparatus  required  is,  in  fact,  less  complex 
than  that  of  a  grain  distillery.  Moreover,  the  working  of 
the  molasses  requires  relatively  little  supervision,  and 
much  less  practical  knowledge  on  the  part  of  the  operators 
than  does  the  distillation  of  grains.  Yet  there  are  few  manu- 
factories which  utilize  rationally  the  former  materials  and 
secure  results  even  approximating  the  theoretical  yield. 

Manufacturers  attribute  the  difficulties  they  encounter 

ss 


FERMENTATION   OF  MOLASSES.  89 

either  to  the  quality  of  molasses  used,  or  to  the  inefficiency 
of  yeasts,  or  to  infection  by  foreign  ferments,  and  they  seek 
to  remedy  these  imperfect  conditions  by  a  strong  acidifica- 
tion of  the  mash.  They  sometimes  try  also  to  regulate  the 
work  by  a  preliminary  heating  of  the  molasses  in  order  to 
eliminate  the  volatile  organic  acids.  These  acids  are  freed 
by  the  addition  of  sulphuric  acid  to  the  mash  at  the  time  of 
acidification. 

The  causes  which  occasion  these  troubles  in  the  working 
of  molasses  are  very  numerous.  We  cannot  here  enter  upon 
a  thorough  study  of  this  question,  but,  nevertheless,  we  think 
we  ought  to  call  the  attention  of  manufacturers  who  are  oc- 
cupied with  fermentations  to  some  of  the  very  frequent 
causes  of  the  smallness  of  the  output,  especially  the  insuffi- 
ciency of  inversion. 

In  the  practice  of  distilling  molasses  this  point  is  wholly 
neglected.  Although  it  is  known  that  non-inverted  sugar  is 
not  fermentable,  little  importance  is  attached  to  the  inversion 
of  the  saccharose  of  molasses,  because  of  the  current  opinion 
that  inversion  is  very  easily  accomplished  owing  to  various 
conditions  of  the  medium. 

If  the  question  is  studied  more  closely,  it  is  seen  that,  on 
the  contrary,  inversion  is  very  slow,  and  that  in  most  cases  it 
is  not  completed  at  the  end  of  the  fermentation. 

The  reason  that  in  practice  little  attention  is  paid  to  hy- 
dration is  that  during  fermentation  two  factors  are  generally 
counted  on :  first,  the  sulphuric  acid  which  has  been  intro- 
duced into  the  molasses  and  which  is  considered  sufficient  in 
itself  to  produce  inversion ;  second,  the  yeast  which  is  sup- 
posed to  be  an  inexhaustible  source  of  sucrase. 

Let  us  see  to  what  extent  each  of  these  two  factors  con- 
tributes to  the  inversion,  and  let  us  first  study  the  part  played 
by  the  acid. 

By  the  addition  of  acid  to  mashes  of  molasses  there  is 
secured  in  practice  an  acidity  corresponding  to  from  1  to  2.5 
grams  per  litre  of  sulphuric  acid. 


go  THE   ENZYMES  AND    THEIR   APPLICATIONS. 

The  acidification  of  the  musts  is  done  at  a  low  or  high 
temperature  according  to  the  distillery. 

To  give  an  idea  of  the  inverting  power  which  these 
amounts  of  acid  possess,  let  us  add  to  a  certain  number  of 
ioo-gram  specimens  of  a  10  per  cent  solution  of  cane-sugar 
different  amounts  of  sulphuric  acid  and  let  us  submit  these 
experiments  for  24  hours  to  a  temperature  of  300. 

Numbers  of                Number  of  grams  of               Grams   of 
the  specimens.           sulphuric   acid   per  litre.         invert-sugar. 
I 2.5 I 

2 5    • 1.8 

3 10 3-3 

4 25 6.7 

Thus  when  we  submit  our  experiment  to  the  action  of  2.5 
grams  of  sulphuric  acid,  the  greatest  amount  used  in  manu- 
facture, we  obtain  at  the  end  of  24  hours  only  10  per  cent  of 
invert-sugar,  and  in  order  to  obtain  67  per  cent,  we  must  use 
25  grams  of  acid,  or  a  quantity  10  times  greater. 

The  action  of  the  cold  acid  is  not  then  an  important  factor 
in  inversion.  The  results  obtained  by  boiling  the  sugar  solu- 
tions appear  quite  different,  it  is  true. 

If,  to  ascertain  the  influence  of  high  temperatures,  we  re- 
peat the  preceding  experiments  at  900,  we  shall  observe  that 
with  the  smallest  quantity  of  acid  (0.5  gr.  per  litre)  we  pro- 
duce a  complete  inversion  of  the  saccharose. 

We  may  conclude  from  this,  that  heating  the  molasses 
with  slight  amounts  of  acid  present  is  very  important  from 
the  point  of  view  of  inversion.  But  molasses  does  not  act 
towards  the  different  factors  in  the  same  manner  as  the  solu- 
tions of  pure  sugar.  In  fact,  the  acidity  found  in  mashes  of 
molasses  comes,  not  from  mineral  acids  which  have  been 
added  to  them,  but  rather  from  organic  acids  which  have 
been  freed  by  the  sulphuric  acid,  and  which  act  on  the  sac- 
charose with  much  less  energy  than  inorganic  acids.     More- 


FERMENTATION  OF  MOLASSES.  91 

over,  the  presence  of  salts  in  the  molasses  weakens  the  action 
of  the  acids. 

The  effect  produced  practically  by  heating  acid  molas- 
ses may  be  shown  by  the  following  experiments:  ioo 
grams  of  molasses  are  diluted  in  400  grams  of  water. 
Different  specimens  of  this  must  are  taken ;  they  are  acidified 
with  varying  amounts  of  sulphuric  acid;  they  are  kept  boil- 
ing for  some  time,  then  cooled  and  brought  up  to  their 
original  volume.  By  examining  the  rotatory  power  of  these 
specimens,  one  may  study  the  course  of  the  transformation  in 
the  presence  of  different  amounts  of  acid.  The  solution  be- 
fore inversion  gives  a  rotation  of  380  to  the  right,  and  after 
complete  inversion  a  rotation  of  SV  to  the  left.  Moreover 
here  are  the  intermediate  results: 

Numbers  of  Grams  of  sulphuric  Rotation  to 

the  specimens.  acid  per  litre.  the  right. 

1 1-25 37° 

2 2.5    36 

3 5      35 

4 10      24 

5 I2-5    3-6 

With  the  amount  of  acid  used  in  manufacture,  or  two 
grams  and  a  half,  inversion  is  then  trifling;  rotation  de- 
creases only  from  380  to  360.  We  find,  indeed,  that  by  using 
five  times  as  much  acid,  we  are  still  far  from  obtaining  com- 
plete inversion;  sulphuric  acid  used  in  an  amount  of  12.5  gr. 
still  gives  us  a  rotation  to  the  right  of  3. 6°,  while  complete  in- 
version would  have  given  a  rotation  to  the  left  of  8.50. 

In  many  manufactories  it  is  the  custom  to  boil  acidified 
molasses  after  diluting  it  with  water.  It  is  seen  that  in  this 
case  practically  no  inversion  takes  place.  We  have  been 
able,  however,  to  ascertain  that  the  inversion  is  hastened 
when  the  acid  molasses  is  heated  before  dilution. 

In  the  practice  of  distilling  molasses  it  is  the  sucrase  of 


92  THE  ENZYMES  AND   THEIR  APPLICATIONS. 

the  yeasts  and  not  the  acids  used  which  produces  hydration 
of  the  saccharose,  and  the  course  of  the  fermentation  de- 
pends, in  very  large  measure,  on  the  manner  in  which  the 
secretion  of  diastases  by  the  cells  is  accomplished.  Now,  the 
action  of  the  sucrase  is  considerably  influenced  by  the  saline 
substances  contained  in  the  molasses.  The  following  experi- 
ment is  well  calculated  to  demonstrate  this  fact : 

'  Acidify  a  sugar  solution  at  120  Balling  with  sulphuric 
acid  in  the  proportion  of  0.5  grams  per  litre,  and  take  from 
this  must  two  specimens,  A  and  B.  To  specimen  A,  which  is 
the  control  specimen,  add  10  cubic  centimetres  of  yeast  su- 
crase. The  second  specimen  receives  the  same  quantity  of 
sucrase,  then  the  accurately  neutralized  ash  of  100  cubic 
centimetres  of  a  must  of  molasses  of  120,  Ball.  The  two 
specimens  are  then  left  in  a  water-bath  at  300. 

Here  is;the  comparative  progress  of  inversion  in  the  two 
specimens : 

Minutes.  A-  B- 

Invert-sugar.  Invert-sugar, 

40  min 4-7% 2.4% 

2  h 5-79   2.9 

3  h 7.0     3.2 

4  h 9.2      4.6 

These  figures  prove  in  a  conclusive  manner  that  the  min- 
eral substances  of  the  molasses  retard  inversion  considerably. 
After  four  hours  of  action,  there  is  found  in  the  control  solu- 
tion 9.2%  of  invert-sugar,  while  in  the  solution  to  which  ash. 
of  molasses  has  been  added  there  is  found  only  4.6%.  These 
data  show  the  nature  of  the  difficulties  encountered  in  the 
fermentation  of  molasses. 

Still  it  may  be  objected  that  in  the  fermentation  indus- 
tries, a  solution  of  diastase  is  not  used :  but  that  inversion  is 
accomplished  by  living  cells.  It  must  then  be  admitted  that 
the  conditions  of  transformation  are  completely  different; 
that  since  inversion  by  yeasts  can  be  accomplished  inside  the 


FERMENTATION   OF  MOLASSES.  93 

cells,  the  composition  of  the  exterior  liquid  naturally  has  a 
much  smaller  influence  on  that  account. 

To  answer  this  objection  we  have  made  the  following  ex- 
periments: 

To  a  10  per  cent  solution  of  cane-sugar  is  added  yeast 
ash.  This  solution  serves  as  the  basis  of  two  specimens,  A 
and  B,  each  of  500  cubic  centimetres.  In  specimen  A  there 
are  introduced  the  neutralized  ash  of  50  grams  of  molasses 
and  5  grams  of  yeast.  Specimen  B  is  submitted  to  the  influ- 
ence of  the  same  quantity  of  yeast,  but  without  the  addition 
of  salts. 

Here  is  the  comparative  course  of  the  fermentation  of  the 

two  specimens : 

A  B 

....  (    Invert-sugar 0.5%      1.8% 

After  6  hours    \     .  ,     ,    ,    fe  ° ' 

(   Alcohol 0.4         0.65 

Ar  ,  (    Invert-sugar 0.2  3 

After   12  hours  <      ■      ,    ,   &  °  , 

{   Alcohol 1.5  2.6 

A  .  ,  (   Invert-sugar 0.5  0.2 

After  24  hours  ■{.,,, 

[  Alcohol 3  5.9 

By  comparing  the  quantity  of  invert-sugar  after  six  hours 
in  the  two  solutions,  we  see  that  inversion  proceeds  much 
more  slowly  in  solution  A,  treated  with  the  salts  contained  in 
the  molasses.  It  is  true  that  after  24  hours  we  find  a 
greater  quantity  of  invert-sugar  in  solution  A  than  in  solu- 
tion B,  but  if  we  take  into  account  the  quantity  of  alcohol 
present  at  this  time  in  the  two  solutions,  it  becomes  evident 
that  hydration  has  followed  a  much  more  regular  course  in  B 
than  in  A. 

Phenomena  of  this  nature — slowness  in  fermentation,  ir- 
regularities in  the  progress  of  the  transformation — are  often 
found  in  molasses  distilleries  and  are  generally  attributed  to 
the  degeneracy  of  the  yeasts. 

This  opinion  is  absolutely  erroneous;  yeast  does  not  gen- 
erally degenerate  in  molasses  mashes ;  on  the  contrary,  it  re- 


94  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

produces  abundantly  and  the  cells  formed  under  these  con- 
ditions generally  have  great  activity.  These  yeasts  cause  a 
very  rapid  fermentation  of  grain  mashes,  but  the  quantity  of 
sucrase  which  they  secrete  diminishes. 

It  is  to  this  diastatic  weakening  that  we  must  attribute 
the  difficulties  encountered  in  fermenting  unchanged  sac- 
charose with  yeasts  cultivated  in  molasses. 

Not  all  the  beer-yeasts  contain  the  same  quantity  of  su- 
crase; the  quantity  of  the  enzyme  secreted  varies  with  the 
special  variety.  In  the  choice  of  a  yeast  for  molasses,  one 
should  first  take  account  of  the  inverting  power,  as  well  as  of 
the  degree  of  resistance  of  the  active  substance  which  it  con- 
tains. Generally  the  distiller  seeks  to  substitute  quantity  for 
quality  in  the  yeasts.  This  practice  is  far  from  rational. 
The  expense  in  yeast  is  thus  rendered  quite  large  and  the 
alcoholic  yield  is  diminished,  for  the  yeast  consumes  a  part 
of  the  carbohydrate  for  the  construction  of  its  tissues  and  for 
their  maintenance. 

To  test  a  yeast  with  a  view  to  its  action  on  molasses  it  is 
not  enough  to  determine  its  inverting  power  in  a  solution  of 
pure  saccharose.  It  is  better  to  make  the  experiment  with 
saline  substances  present.  The  advantage  of  this  method  is 
that  it  gives  more  certain  results,  because  it  approximates 
actual  industrial  conditions.  We  have  had  occasion  to  make 
experiments  with  yeasts  from  different  sources,  and  these  ex- 
periments have  shown  us  that  the  degree  of  resistance  of  the 
sucrase  contained  in  the  cells  differs  much  according  to  the 
variety.  These  experiments  have  proved  to  us,  moreover, 
that  the  resistance  of  the  enzyme  plays  a  very  important  part 
as  influencing  the  yield. 

Compressed  yeasts  as  well  as  beer-yeasts  have  been  re- 
placed in  the  fermentation  of  molasses  by  leaven.  The  dis- 
tiller cultivates  his  yeasts  himself  and  for  this  purpose  uses 
mashes  of  grain  prepared  either  by  sulphuric  acid  or  by  malt. 

There  is  generally  used  for  the  preparation  of  the  mash 


FERMENTATION   OF  MOLASSES.  95 

leaven  3  to  5  kilograms  of  grain  for  100  kilograms  of 
molasses.  In  many  distilleries  this  amount  of  grain  is  in- 
creased. Sometimes  it  is  thought  best  to  add  to  the  molasses 
or  to  the  yeast  mash  a  certain  amount  of  nutritive  nitroge- 
nous materials,  such  as  rootlets  of  malt,  amides,  and  pep- 
tones. 

Undoubtedly  the  use  of  grains  and  nitrogenous  materials 
furnishes  appreciable  results  with  certain  varieties  of  yeasts 
which  demand  a  special  medium  to  acquire  their  inverting 
power.  It  must,  however,  be  recognized  that  the  very  prin- 
ciple of  this  practice  is  wholly  false  and  that  the  results  ob- 
tained are  far  from  being  satisfactory  from  an  economic  point 
of  view.  Molasses  contains  all  the  nutritive  substances 
necessary  to  feed  the  yeast-cells  abundantly. 

If  a  variety  of  yeast  cannot  become  accustomed  to  acting 
upon  molasses,  if  a  special  manner  of  nutrition  must  be 
adopted  so  that  it  can  live  in  this  medium,  this  yeast  should 
be  given  up  and  another  less  delicate  kind  should  be  used. 

While  visiting  the  molasses  distilleries  at  Breslau,  Leip- 
zig, Darmstadt,  etc.,  in  1895.  we  found  out  that  the  leaven 
used  for  the  fermentation  of  molasses  in  these  manufactories 
cost  from  8  to  10  francs  per  hundred  litres  of  alcohol  manu- 
factured. This  expense,  a  total  loss,  arose  from  the  fact  that 
the  distilleries  used,  for  the  making  of  their  leaven,  malt  and 
grains  without  which  their  yeasts  did  not  work  well.  We 
advised  them  to  take  a  yeast  suitable  for  action  upon  molas- 
ses, and  we  have  since  had  the  satisfaction  of  learning  of  the 
almost  complete  suppression  of  the  use  of  grain  for  leaven 
in  those  places. 

The  leaven  is  at  present  made  with  pure  molasses  and  the 
yield  in  alcohol  is  unquestionably  greater. 

Most  kinds  of  beer-yeast  furnish  a  sucrase  of  little  resist- 
ance, but  their  greater  or  less  alterability  depends  especially 
on  the  culture  medium  in  which  they  are  developed.  Sucrase 
secreted  by  yeasts  cultivated  in  molasses  possesses  a  resist- 


96  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

ance  inferior  to  that  of  the  same  yeasts  cultiyatecl  in  a  must  of 
grains  or  malt. 

This  weakening  of  the  resistance  is  due  neither  to  the 
nature  of  the  enzyme  secreted  nor  to  the  prolonged  contact 
with  saline  substances ;  its  true  cause  is  rather  the  sudden  pas- 
sage of  the  cells  from  one  medium  to  another. 

We  have  found  that  yeasts  capable  of  producing  the  fer- 
mentation of  dilute  molasses  can  be  brought  to  effect  the 
complete  fermentation  of  very  concentrated  mashes  by  ac- 
climating these  yeasts  to  the  new  medium  by  gradually  fur- 
nishing them  with  solutions  of  increasing  concentration. 

The  changes  which  are  produced  in  the  resistance  of  a 
sucrase  by  acclimatization  to  the  medium  may  be  demon- 
strated by  the  following  experiments: 

.  Sucrase  is  extracted  from  yeasts  in  different  stages  of 
habituation  and  tried  simultaneously  on  a  solution  of  pure 
sugar,  and  on  a  solution  of  sugar  to  which  ash  of  molasses 
has  been  added.  It  is  thus  found  that  the  yeast  has  acquired 
new  properties  and  furnishes  an  enzyme  which  is  little 
changed  when  saline  substances  are  present.  These  new 
properties  acquired  by  the  yeasts  are,  however,  transitory 
properties.  * 

*  These  experiments  throw  a  special  light  on  the  mechanism  of  ac- 
climatization as  well  as  on  the  individuality  of  diastases. 

By  studying  the  sensitiveness  of  beer-yeasts  to  the  action  of  different 
antiseptics,  we  have  established  the  fact  that  one  can  acclimatize  the  yeasts 
to  relatively  large  amounts  of  these  agents.  Thus,  a  beer-yeast  which 
shows  itself  very  sensitive  to  the  action  of  10  milligrams  of  hydrofluoric 
acid  and  which  no  longer  gives  any  fermentation  in  the  nutritive  mash 
can  be  made  to  reproduce  in  the  presence  of  30  times  as  much  of  this 
acid,  and  to  give  rise  to  very  active  fermentation.  This  acclimatization 
requires  that  one  accustom  the  yeast  to  increasing  amounts  of  this  anti- 
septic. 

The  cells  thus  obtained  acquire  characteristic  properties: 

The  fermenting  power  is  considerably  augmented,  while  the  power  of 
multiplication  is  reduced  to  its  lowest  limits. 

Yeast  acclimated  to  antiseptics  preserves  the  characteristic  property 
of  resisting  them  for  several  months,  even  when  it  is  daily  cultivated  in 
solutions  free  from  the  agent  to  which  they  have  become  accustomed. 


FERMENTATION   OF  MOLASSES.  97 

The  difficulty  of  inversion  in  the  fermentation  of  molasses 
may  also  arise  from  some  other  cause  than  the  insufficiency 
of  the  sucrase  of  the  yeast.  Thus,  strong  acidity  of  the 
musts  or  a  great  concentration  of  the  sugar  may  produce  a 
slackening"  in  the  fermentation. 

The  excess  of  sugar  acts  unfavorably  on  account  of  the 
accumulation  of  alcohol  in  the  musts.  Even  with  5  per  cent 
of  alcohol  present,  the  diastatic  force  is  influenced,  and  with 
10  per  cent  the  inversion  proceeds  very  slowly  indeed. 

The  acidity.of  the  musts,  such  as  is  found  in  practice,  does 
not  act  directly  on  the  sucrase  and  the  retardation  observed 
must  rather  be  attributed  to  foreign  ferments,  which,  favored 
by  the  acidity,  develop  in  the  mash. 

From  molasses  which  is  fermentable  with  difficulty  we 
have  isolated  bacteria  which  produced  a   slight  acidity  in 


Acclimatization  has  then  produced  a  profound  change  in  the  cells,  and  one 
which  is  transmitted  from  one  generation  to  another. 

Quite  a  different  thing  is  observed  in  the  action  of  saline  substances  on 
yeast. 

The  resistance  which  sucrase  acquires  by  acclimatization  commences 
to  be  weakened  as  soon  as  the  yeast  is  again  found  in  a  medium  free  from 
salts. 

The  properties  of  sucrase  are  here  then  closely  related  to  the  composi- 
tion of  the  medium.  This  fact  contradicts  the  hypothesis  of  the  existence 
of  different  enzymes  acting  on  the  same  body  and  producing  the  same 
chemical  reactions. 

According  to  this  hypothesis,  indeed,  there  exist  different  sucrases. 
•The  sucrase  of  Aspergillus  nigcr,  for  example,  would  be  a  different  sub- 
stance from  the  sucrase  of  yeast,  etc.  One  would  also  have  to  distinguish 
between  the  sucrases  of  different  yeasts,  because  these  are  not  equally 
sensitive  to  the  temperature  and  to  the  reaction  of  the  medium.  The 
variable  resistance  of  sucrase  to  chemical  agents  would  evidently  lead  to 
new  distinctions,  but  these  latter  distinctions  would  be  absolutely  illusory. 

The  difference  in  the  manner  of  action  in  the  presence  of  chemical  sub- 
stances arises,  not  from  a  change  in  the  nature  of  the  sucrase,  but  rather 
from  difference  in  the  external  media. 

The  same  thing  must  occur  with  sucrases  of  different  origin,  showing- 
different  properties. 

In  fact,  the  diastase  is  accompanied  in  the  cells  which  secrete  it  by 
different  substances  which  modify  its  properties. 


98  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

sugar  solutions.  Alcoholic  fermentation  is  manifestly  ar- 
rested by  such  ferments.  These  micro-organisms  act  also  on 
yeast  in  grain  mashes,  but  they  are  shown  to  be  specially  dan- 
gerous in  the  fermentation  of  saccharose. 

In  the  presence  of  these  organisms  the  fermentation  of 
the  sugar  solutions  is  arrested  when  the  must  has  still  only 
a  slight  acidity  and  a  small  quantity  of  sugar  has  as  yet  been 
transformed. 

The  difficulties  in  fermentation  of  molasses  caused  by  lack 
of  sucrase  are  accompanied  by  the  following  symptoms : 

The  fermentation  begins  regularly,  but  when  about  50 
per  cent  of  the  sugar  is  transformed,  that  is,  before  the  end 
of  the  principal  fermentation,  there  is  suddenly  found  a 
noticeable  slackening,  then  a  stop  which  is  prolonged  for 
several  hours.  The  yeast  is  deposited  slowly ;  there  is  pro- 
duced little  by  little  new  amounts  of  sucrase  and  the  fermen- 
tation recommences,  often  with  great  energy.  Then  a  second 
stop  occurs  which  is  generally  final.  The  mash  in  fermenta- 
tion often  contains  at  this  period  considerable  quantities  of 
non-inverted  sugar.  Such  is  the  course  of  events  when  the 
work  is  carried  on  in  the  presence  of  antiseptics. 

In  the  opposite  case,  the  fermentation  has  an  entirely  dif- 
ferent aspect. 

When  the  slackening  occurs,  the  mash,  invaded  by 
bacteria,  becomes  decidedly  acid.  The  yeast  degenerates  and 
the  fermentation,  once  stopped,  does  not  recommence,  or  at 
least,  does  so  only  very  feebly. 

From  the  practical  point  of  view,  it  is  well  for  the  distiller 
of  molasses  to  pay  more  attention  than  he  usually  does  to  the 
manner  of  inversion  of  cane-sugar  during  fermentation.  It 
is  especially  necessary  to  exercise  great  care  in  the  choice  of 
yeasts.  It  is  then  indispensable  to  protect  the  mashes  by 
antiseptics  against  foreign  ferments.  It  is  also  well  to  filter 
or  decant  the  molasses  mashes  after  their  acidification.  Not 
all  the  ferments  are  destroyed  by  simply  boiling.  The 
bacteria  found  in  difficultly  fermentable  molasses  are  only 


FERMENTATION  OF  MOLASSES.  99 

destroyed  at  a  temperature  of  no°,  though  they  may  be 
easily  removed  either  by  filtration  or  by  decantation. 

BIBLIOGRAPHY. 


Effront.— Etude  sur  la  fermentation  des  melasses.     Moniteur  scientifique, 

1894 

Bui 

1894 


189.4,  p.  461.  .  . 

Bulletin  de  la  Societe   d'encouragement  pour  1  Industrie  national 


CHAPTER  VIII. 

AMYLASE. 

Presence  of  amylase  in  vegetable  and  animal  cells. — Preparation. — Cohn- 
heim's  method. — Lintner's  method. — Effront's  method. — Wroblew- 
sky's  method. — Properties. — Influence  of  quantity,  time,  and  tempera- 
ture.— Influence  of  chemical  agents;  acids,  alkalies,  salts. — Substances 
which  accelerate  diastatic  action. 

The  enzyme  called  amylase,  or  simply  diastase,  is  a  solu- 
ble ferment  hydrating  starch  and  transforming  it  into  mal- 
tose and  dextrins. 

The  existence  of  this  enzyme  was  first  observed  by 
Kirchoff,  in  1814,  in  gluten.  Dubrunfaut,  Pay  en,  and  Persoz 
have  since  studied  this  substance  thoroughly. 

Amylase  is  widely  distributed  in  nature.  It  is  found  in 
barley,  oats,  rice,  maize,  and  in  general,  in  all  cereals. 

The  raw  grains  have  little  amylase,  the  enzyme  being 
formed  especially  in  the  course  of  germination. 

The  presence  of  amylase  has  been  observed  in  the  tubers 
of  potatoes,  as  well  as  in  the  leaves  and  shoots  of  different 
plants. 

The  transformation  of  starch  into  carbohydrates  assimi- 
lable by  living  cells  being  generally  accompanied  by  the  ac- 
tion of  amylase,  it  may  be  believed  that  this  substance  plays 
a  very  important  part  in  the  formation  of  vegetable  tissues. 
Yet  the  transformation  of  starch  is  not  always  produced  by 
the  aid  of  amylase,  and  because  a  cell  brings  about  the 
transformation  of  amylaceous  materials  does  not  at  all  prove 
that  it  secretes  this  diastase. 

We  shall  see  later  that  other  enzymes  exist  which  act  on 


AMYLASE.  ioi 

starch  and  render  it  assimilable  and  suitable  for  the  construc- 
tion of  tissues. 

Wortmann  asserted  that  the  assimilation  of  starch  is  not 
always  accompanied  by  the  action  of  enzymes,  and  he  be- 
lieved that  he  demonstrated  that  protoplasm  alone  can  by 
itself  produce  a  hydrating  and  dissolving  action  on  starch. 
This  view  may  be  seriously  questioned,  however.  It  is  true 
that  in  the  leaves  of  plants  where  a  very  active  transforma- 
tion of  starch  is  brought  about,  there  are  generally  found 
quantities  of  diastase  small  in  comparison  with  the  work  ob- 
served. It  is  equally  true  that  often  the  stalks  and  stems  do 
not  secrete  active  substances,  while  in  these  organs  is  found 
an  energetic  assimilation  of  starch.  But  these  facts  are  not 
sufficient  to  prove  the  direct  action  of  protoplasm  in  the  hy- 
dration of  starch.  The  fact  that  amylase  has  not  been  found 
in  the  cells  may  simply  arise  from  difficulties  like  those  which 
occur  in  the  study  of  sucrase ;  in  other  words,  amylase  may 
be  more  or  less  retained  within  the  cells,  or  it  may  enter  into 
combination  with  other  substances  and  thus  become  more  or 
less  soluble. 

As  we  have  found  for  tannin,  amylase  is  sometimes 
present  in  an  inactive  form,  because  the  environmental 
conditions  under  which  it  is  found  are  unfavorable  for 
its  action.  In  this  case,  it  again  acquires  its  normal  prop- 
erties as  soon  as  it  is  placed  under  favorable  conditions.  We 
know,  moreover,  that  the  effect  produced  by  an  enzyme  de- 
pends especially  upon  the  conditions  of  the  medium,  and  we 
have  reason  to  believe  that  in  living  cells  the  action  of  dia- 
stases is  more  energetic  than  in  our  experiments.  It  is  prob- 
able, moreover,  that  with  a  very  small  quantity  of  active  sub- 
stance one  could  obtain  marked  results  if  the  conditions  of 
the  medium  are  favorable.  If,  then,  amylase  has  not  been 
found  in  the  various  vegetable  organs  examined,  these  nega- 
tive results  may  be  attributed  to  the  different  circumstances 
which  we  have  just  enumerated.  Then  it  is  not  at  all  estab- 
lished that  protoplasm  would  be  capable,  without  the  inter- 


IQ2  THE  ENZYMES  AND    THEIR   APPLICATIONS. 

vention  of  enzymes,  of  hydrating  starch,  and  all  the  data 
which  we  possess  on  enzymes  tends  rather  to  prove  that  dia- 
static  action  takes  place  here  also. 

Again,  amylase  is  found,  according  to  some  authors,  in 
moulds.  Thus,  Aspergillus  niger,  or  Penicillium  glaucum,  if 
cultivated  under  certain  conditions,  would  secrete  a  certain 
quantity  of  amylase ;  the  presence  of  this  diastase  in  moulds 
is  very  rare,  however.  The  diastase  which  hydrates  starch  is. 
met  with,  not  only  in  the  vegetable  kingdom,  but  also  in 
animal  secretions.  It  is  present  in  the  saliva,  the  pancreatic 
juice,  and  the  liver.  The  constant,  presence  of  the  active 
substance  in  the  saliva  may  be  explained  by  two  different 
theories ;  first,  that  amylase  is  secreted  by  the  salivary 
glands ;  or,  second,  that  it  is  due  to  organized  ferments  which 
are  found  in  the  mouth,  and  feed  upon  amylaceous  materials. 
Claude  Bernard,  who  studied  this  question,  was  a  supporter 
of  the  latter  theory.  By  heating  saliva  to  ioo°,  he  found  the 
complete  destruction  of  the  active  substance.  This  saliva, 
having  lost  the  property  of  acting  on  starch,  became  active 
when  left  for  some  time  at  the  ordinary  temperature.  He 
attributes  this  phenomenon  to  the  development  in  the  saliva 
of  new  ferments  capable  of  furnishing  to  the  surrounding 
liquid  new  quantities  of  diastase.  The  appearance  of  the  dia- 
stase could,  however,  be  explained  by  the  action  of  the  tem- 
perature on  the  ferments  of  the  saliva.  Certain  cells  which 
have  not  been  destroyed  by  the  heat  might  still  retain  amy- 
lase, and  the  enzyme,  when  the  temperature  is  lowered, 
would  diffuse  into  the  liquid.  However  this  may  be,  the  ap- 
pearance of  amylase  in  the  saliva  may  be  explained  otherwise 
than  by  a  secretion  of  the  glands. 

To  settle  this  question  definitely,  the  experiment  of 
Claude  Bernard  must  be  repeated  under  such  conditions  that 
the  action  of  any  organized  ferments  can  be  avoided. 

Preparation  of  Amylase. — Amylase  can  be  precipitated 
from  its  solutions,  either  by  mechanical  precipitation  or  by 
the  action  of  alcohol.     According  to  Cohnheim,  the  diastase 


AMYLASE.  103 

can  be  extracted  from  the  saliva  by  the  following  method: 
quicken  salivation  by  rinsing-  the  mouth  with  ether.  The 
saliva  is  then  collected  and  a  slight  amount  of  phosphoric 
acid  is  added.  The  acid  liquid  is  then  neutralized  with  great 
care  by  the  aid  of  very  dilute  lime-water.  Thus  calcium 
phosphate  is  formed,  which  carries  down  the  diastase  as  well 
as  other  nitrogenous  materials.  This  precipitate  is  removed 
by  filtration,  then  washed  on  the  filter  with  a  volume  of  water 
equal  to  that  of  the  saliva  used.  By  the  washing,  the  dia- 
stase goes  into  solution.  It  is  precipitated  from  this  solution 
by  a  suitable  addition  of  alcohol.  The  preparation  of  amy- 
lase is  accomplished  much  more  easily  by  the  use  of  an  in- 
fusion of  malt.  Payen  was  the  first  to  find  that  the  active 
substances  of  an  infusion  can  be  precipitated  from  the  solu- 
tion by  a  suitable  addition  of  alcohol.  The  product  thus 
obtained  is  unfortunately  far  from  pure;  furthermore,  it 
changes  readily  in  the  air,  oxidizes,  and  becomes  inactive 
very  quickly,  and  assumes  a  very  dark  color. 

The  alteration  of  amylase  is  facilitated  by  foreign  sub- 
stances precipitated  at  the  same  time  as  the  diastase  by  the 
alcohol.  Different  means  have  been  proposed  to  avoid 
these  difficulties.  Payen  and  Persoz,  for  example,  advise 
adding  to  the  infusion  of  malt  a  quantity  of  alcohol  insuffi- 
cient to  precipitate  the  diastase,  then  bringing  the  alcoholic 
solution  to  a  temperature  of  700,  which,  according  to  them, 
would  cause  the  coagulation  of  foreign  substances,  especially 
albuminoid  matter.  This  operation  finished,  the  coagulated 
substances  are  separated  from  the  liquid  which  contains  them 
and  an  excess  of  alcohol  is  added  to  cause  a  precipitation  of 
the  amylase.  By  this  method  there  is  obtained  a  white  prod- 
uct which  is  not  very  changeable  but  which  possesses  only 
a  very  slight  activity. 

Much  more  satisfactory  results  are  obtained  by  Lintner's 
method :  One  part  of  finely  grouwl  malt  is  mixed  with  four 
parts  of  20  per  cent  alcohol ;  it  is  allowed  to  stand  for  24 


104  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

hours ;  then  the  liquid  is  poured  off  from  the  malt,  and  fil- 
tered, and  to  each  volume  of  filtered  liquid  2  volumes  of  ab- 
solute alcohol  is  added.  Thus  a  flocculent  precipitate  is 
formed;  the  clear  solution  is  decanted  and  the  precipitate 
collected  on  a  filter.  After  a  first  washing  with  alcohol  and 
ether,  the  precipitate  is  ground  in  a  small  mortar  with  a  little 
alcohol ;  it  is  then  replaced  on  the  filter  and  washed  a  second 
time  with  alcohol  and  ether,  then  dried  in  vacuo.  By  this 
method  is  obtained  a  product  which  Lintner  calls  raw  dia- 
stase and  which  can  be  still  further  purified  by  solution  in 
water  and  precipitation  by  alcohol.  This  purification  leads 
to  a  product  of  constant  composition  but  of  slight  activity. 

This  method  gives  good  results  if  the  operations  are  per- 
formed rapidly,  in  order  to  prevent  the  precipitated  diastase 
irom  coming  in  contact  with  the  air  before  it  is  completely 
dehydrated.  Nevertheless,  the  product  obtained  is  very  rich 
in  ash  as  well  as  foreign  materials  which  have  been  precipi- 
tated from  the  infusion  of  malt  by  the  alcohol. 

To  obtain  purer  and  more  active  products  we  advise 
using  another  method. 

To  diminish  the  quantity  of  extractive  substances  in  the 
infusion  of  malt  which  do  not  possess  diastatic  power,  fer- 
ment this  infusion  with  yeasts  which  have  been  previously 
limited  to  a  scant  supply  of  nitrogen.  The  alcoholic  fermen- 
tation, caused  by  these  yeasts  in  the  infusion  of  malt, 
destroys  a  great  part  of  the  carbohydrates,  eliminates  a  con- 
siderable quantity  of  albuminoid  matter  and  salts,  but  leaves 
the  diastase  absolutely  intact.  This  is  accomplished  as  fol- 
lows :  macerate  100  grams  of  malt  reduced  to  powder  with 
300  grams  of  water  at  a  temperature  of  30°  for  18  hours.  Stir 
the  mixture  every  half  hour.  The  mass  freed  from  the  liquid 
T^y  pressure  is  thoroughly  washed  with  water, — which,  to- 
gether with  the  original  extract,  is  filtered.  The  filtrate  is 
made  up  to  300  cubic  centimetres,  10  grams  of  beer-yeast 
added  and  left  at  a  temperature  of  280  for  48  hours.  It  is  then 
filtered,  and  to  the  clear  liquid  is  added  700  cubic  centimetres 


AMYLASE.  105 

of  alcohol.  The  yeast  used  for  this  preparation  must  first  of 
all  have  remained  for  24  hours  in  a  10  per  cent  solution 
of  sugar.  The  fermentation  causes  the  yeast  to  lose  a  part 
of  its  nitrogen  and  gives  it  an  avidity  for  albuminoid 
materials.  With  100  grams  of  malt  we  have  obtained  from  3 
to  3.5  grams  of  a  white  substance  having  the  same  activity  as 
80  grams  of  the  malt  used. 

A  new  method  of  preparation  of  diastase  has  been  re- 
cently proposed  by  Wroblewsky,  who  considers  that  diastase 
prepared  by  the  ordinary  methods  is  always  found  mixed 
with  a  pentose,  arabinose.  The  method  consists  of  a  par- 
tial precipitation  caused  by  the  action  of  salts. 

The  author  adds,  drop  by  drop,  to  a  solution  of  amylase, 
ammonium  sulphate  until  a  turbidity  is  produced  in  the  solu- 
tion. Then  the  liquid  contains  50  per  cent  of  ammonium 
sulphate;  allowed  to  stand  for  some  time  there  is  produced 
a  yellowish  flaky  precipitate,  which  is  separated  and  washed 
with  a  54  per  cent  solution  of  ammonium  sulphate.  This 
precipitate  is  very  active:  added  to  a  solution  of  starch,  it 
completely  and  almost  instantaneously  transforms  it  into 
sugar.  According  to  the  author  this  deposit  would  be  com- 
posed of  pure  diastase. 

To  the  liquid  from  which  the  precipitate  has  been  separ- 
ated is  added  again  ammonium  sulphate  up  to  the  amount  of 
60  per  cent ;  a  new  deposit  is  then  produced  which,  separ- 
ated, washed,  and  examined,  has  been  recognized  as  being 
a  mixture  of  a  pentose,  arabinose,  and  diastase.  Finally,  in  a 
third  operation  the  second  liquid  separated  from  its  precipi- 
tate is  again  taken,  saturated  with  ammonium  sulphate  and 
a  new  product  obtained  composed  wholly  of  the  pentose. 
When  one  wishes  to  obtain  a  very  active  product  then,  one 
must  take  the  first  precipitate  obtained  in  the  solution  con- 
taining 50  per  cent  of  ammonium  sulphate. 

Amylase  prepared  in  this  manner  is  very  soluble  in  water. 
It  is  not  coagulated  by  heating,  either  in  a  neutral  solution,, 
or  after  acidification  with  acetic  acid  or  slight  amounts  of 


106  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

hydrochloric  acid.  A  great  addition  of  hydrochloric  acid, 
however,  produces,  by  heating,  a  coagulation  in  the  form  of 
light  flakes.  The  solution  of  amylase,  with  a  certain  amount 
of  nitric  acid  added,  gives  a  light  precipitate  which  is  redis- 
solved  in  an  excess  of  the  reagent.  It  gives  Millon's  reac- 
tion, also  the  biuret  and  xanthoproteic  reactions.  Its  solu- 
tion gives  a  light  precipitate  with  mercuric  chloride.  Tannic 
acid  added  to  the  solution  of  amylase  produces  a  voluminous 
precipitate  soluble  in  dilute  soda.  This  alkaline  solution  be- 
comes somewhat  discolored  when  left  to  the  air  at  500,  but 
does  not  entirely  lose  its  hydrating  power. 

The  amylase  obtained  by  Wroblewsky  gave  on  analysis 
16.53  Per  cent  °f  nitrogen. 

Properties  of  Amylase. — Amylase  is  endowed  with  two 
distinct  properties :  it  liquefies  starch,  and  transforms  starch, 
as  well  as  dextrin,  into  maltose. 

The  two  properties  of  this  diastase  may  easily  be  shown 
t>y  the  following  experiments: 

To  100  cubic  centimetres  of  water  kept  at  the  boiling 
point  add  10  grams  of  potato-flour  diluted  in  20  cubic  cen- 
timetres of  tepid  water.  The  mixture  forms  a  thick  paste 
which  acquires  still  more  resistance  when  it  is  kept  for  some 
time  in  the  neighborhood  of  ioo°.  To  this  starch  is  added 
several  cubic  centimetres  of  an  infusion  of  malt  and  the 
whole  is  left  in  a  water-bath  at  a  temperature  of  70°-75°.  The 
pasty  mass  quickly  becomes  fluid,  and  in  a  longer  or  shorter 
time,  according  to  the  diastatic  power  of  the  infusion,  the 
starch  is  transformed  into  a  transparent  liquid  passing 
through  filter  paper.  This  liquid,  of  an  insipid  taste,  con- 
tains dextrins  and  traces  only  of  sugar.  With  tincture  of 
iodine  it  takes  a  deep-blue  color.  This  solution  of  dextrins 
is  cooled,  a  little  more  of  the  infusion  added,  and  is  left  to  act 
at  a  temperature  of  50°-6o°.  If  samples  of  this  are  taken 
from  time  to  time  and  analyzed,  it  is  found  that  the  dextrin 
gradually  disappears  and  there  appears  in  the  liquid  a  re- 
ducing sugar :  maltose.     The  successive  changes  which  oc- 


AMYLASE.  107 

cur  in  the  mixture  of  dextrins,  under  the  action  of  the  in- 
fusion of  malt,  may  be  easily  followed  by  the  aid  of  tincture 
of  iodine.  The  deep  blue  color,  obtained  in  the  solution  of 
starch  by  the  iodine,  is  gradually  weakened  as  the  saccharin- 
cation  proceeds.  Several  hues  are  obtained  in  the  course  of 
the  saccharification.  From  the  deep  blue  which  the  starch 
gives  we  pass  to  violet,  then  to  red,  then  to  yellow.  Finally, 
when  the  saccharification  is  well  advanced,  the  iodine  no 
longer  causes  any  coloration. 

The  action  of  the  amylase,  at  the  same  time  saccharifying 
and  liquefying,  has  raised  a  doubt  as  to  the  individuality  of 
this  enzyme.  The  hypothesis  has  been  suggested  that  two 
different  enzymes  are  present,  because  the  two  diastatic 
functions  of  amylase  appear  at  very  different  temperatures 
and  because  the  saccharification  and  liquefaction  are  very 
differently  influenced  by  the  chemical  and  physical  conditions 
of  the  medium.  Still  we  must  discard,  at  least  until  it  is  es- 
tablished by  sure  proofs,  this  interpretation  which  brings  a 
new  complication  to  the  study  of  amylase.  This  hypothesis 
would  only  be  acceptable  if  one  could  isolate  completely  each 
of  the  functions  of  amylase,  that  is  to  say  obtain  two  prod- 
ucts, the  one  having  simply  the  liquefying  power,  the  other 
simply  the  saccharifying  power.  But  this  separation  has 
never  been  made.  By  keeping  the  malt  infusion  at  yo°  one 
specially  favors  the  liquefaction,  but  the  product  obtained 
also  contains  a  slight  quantity  of  sugar.  If,  on  the  contrary, 
one  maintains  the  temperature  in  such  a  way  that  saccharifi- 
cation is  favored,  that  is  at  50°-6o°,  a  slight  liquefaction  is 
produced  at  the  same  time. 

Influence  of  Quantities. — The  study  of  the  action  of  amy- 
lase leads  to  conclusions  analogous  to  those  furnished  by  the 
study  of  the  mode  of  action  of  sucrase.  It  is  found  in  fact 
if  the  course  of  saccharification  is  followed,  that  the  quantity 
of  sugar  formed  at  the  beginning  of  hydration  is  propor- 
tional to  the  quantity  of  the  enzyme  used.  Afterwards,  when 
the  decomposition  of  the  starch  is  more  advanced,  this  pror 


lo8  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

portionality  ceases  to  exist.  The  course  of  the  saccharifica- 
tion  when,  for  the  same  quantity  of  starch,  increasing  quan- 
tities of  amylase  are  used,  is  shown  by  the  following  table: 

Quantity  of  infusion                                             Quantity  of  maltose 
of  malt  used.                                                           produced. 
I     CC O.  I 

3       0.31 

5       °-49 

10 0.82 

15     I-I 

20       I.I 

30         1.2 

By  checking  the  action  of  the  diastase  after  one  hour,  in 
a  saccharification  made  at  500  with  different  amounts  of  amy- 
lase, it  is  found  that  with  3  cubic  centimetres  of  an  infusion 
of  malt  there  is  obtained  fully  3  times  as  much  maltose  as 
with  1  cubic  centimetre.  If  the  amount  of  diastase  is  still 
further  increased,  there  is  observed  an  increase  in  the  quan- 
tity of  maltose  formed,  an  increase  which  is,  however,  less 
and  less  regular.  Beyond  a  certain  limit  the  quantity  of  the 
infusion  no  longer  influences  the  progress  of  saccharification, 
even  though  at  that  moment  not  all  the  dextrins  contained  in 
the  solution  have  been  transformed. 

In  inversion  by  sucrase  we  have  observed  an  analogous 
course.  Still  the  analogy  is  not  complete.  With  amylase 
the  proportion  is  maintained  until  nearly  40  per  cent  of  the 
starch  is  transformed,  while  with  sucrase  the  proportionality 
is  no  longer  shown  when  15  per  cent  of  the  sugar  is  inverted. 
Moreover,  the  slackening  at  the  end  of  the  action  is  much 
more  pronounced  in  the  case  of  amylase  than  in  that  of  su- 
crase. 

Influence  of  Time. — When  one  studies  the  effect  of  time 
on  the  progress  of  saccharification,  one  finds,  as  before,  that 
at  the  beginning  of  the  action  a  constant  ratio  exists,  and 


AMYLASE.  109 

then  a  slackening  which  is  more  and  more  marked  as  the 
transformation  of  starch  advances.  To  show  the  influence 
of  time  we  cause  a  slight  quantity  of  diastase  to  act  upon  1 
per  cent  starch ;  we  take  samples  from  time  to  time  and  de- 
termine the  quantity  of  sugar  formed. 

The  following  are  the  results  obtained  at  a  temperature 
of  500 : 

Numbers  of  'he  Duration  of  action  Maltose 

samples.  in  minutes.  produced. 

1 15    0-05  gr. 

2 30    0.097 

3 60    0.21 

4 120   0.39 

5 240   0.63 

6 480   0.82 

In  the  four  first  samples  the  quantity  of  maltose  formed  is 
almost  proportional  to  the  duration  of  the  action.  In  the 
others  the  proportionality  ceases  to  exist  and  it  is  interesting 
to  observe  that  when  the  slackening  commences  there  is 
formed  in  the  liquid  nearly  40  per  cent  of  maltose.  It  is  the 
ratio  between  the  quantities  of  transformed  and  non-trans- 
formed product  which  influences  the  progress  of  hydration. 
It  is  this  ratio  which  determines  the  cessation  in  the  propor- 
tionality. 

If,  instead  of  using,  as  we  have  just  done,  a  very  slight 
quantity  of  diastase,  we  repeat  the  experiment  with  double 
amounts  of  infusion,  while  employing  the  same  quantity  of 
starch,  we  find  that  the  constant  ratio  ceases  after  the  first 
hour  of  action.  If  the  quantity  of  diastase  is  still  further  in- 
creased, the  course  of  the  transformation  becomes  irregular 
after  some  minutes. 

Influence  of  Temperature. — By  saccharifying  starch  with 
infusions  of  malt  at  different  temperatures  for  15  minutes,. 
Kjeldahl  has  obtained  the  following  results: 


Ho  THE  ENZYMES  AND    THEIR   APPLICATIONS. 

Temperature.  Reducing  powers. 

18.5   i7-5 

35      30.5 

54      4i-5 

63      42 

66.5    34 

68      29 

70      18 

The  action  of  amylase  is  very  slow  at  o°.  Towards  300 
saccharification  begins  to  progress  rapidly  and  the  activity 
of  the  enzyme  then  increases  very  rapidly  in  intensity  up  to 
6o°.  Above  6o°  the  production  of  maltose  diminishes,  and 
at  a  temperature  of  700,  which  is  the  most  favorable  tem- 
perature for  liquefaction,  the  quantity  of  sugar  produced  be- 
comes insignificant. 

Kjeldahl  and  Bourquelot  have  observed  that  amylase, 
kept  for  some  time  at  temperatures  higher  than  6o°,  acts 
differently  from  a  diastase  that  has  not  been  heated.  An 
infusion  of  malt  kept  for  10  minutes  at  different  temperatures 
and  then  introduced  in  starch  at  500  produces  very  different 
reactions. 

Infusion  of  malt     t  at  630  furnishes  63^  maltose,  37$  dextrin, 
heated    for  10    -j  at  68°         "  35$         "         65$         " 

minutes  '  at  70°         "  17.4$     "         82.6$      " 

The  temperature  to  which  the  infusion  has  previously 
been  brought  has  then  produced  a  change  in  the  mode  of  ac- 
tion of  the  amylase.  The  heated  diastase  causes  a  decom- 
position of  starch  according  to  equations  which  differ  ac- 
cording to  the  temperatures  to  which  the  infusion  has  been, 
brought. 

To  show  the  influence  of  temperature  on  the  progress  of 
saccharification,  the  following  experiment  may  also  be 
made: 

Heat  an  infusion  of  malt  for  12  hours  at  68°,  then 
try  its  fermenting  power  by  comparing  it  with  that  of  the 
same  infusion  not  heated.     At  500  allow  10  cubic  centime- 


AMYLASE.  1.1 1 

tres  of  non-heated  infusion  and  10  cubic  centimetres  of  the 
same  infusion  previously  heated  to  68°  to  act  on  I  per  cent 
starch. 

In  the  first  case  0.6  of  maltose  is  obtained  and  in  the 
second  0.3.  The  diastatic  power  has  then  diminished  by 
half.  Now  the  power  of  the  infusion  heated  to  68°  may  be 
tried  in  starch  of  different  degrees  of  concentration : 

10  c.c.  of  infusion  have  given  in  a  1%  starch 0.3  maltose. 

2%       "         0.6 

3%     "        0.9 

In  2  per  cent  starch  the  heated  infusion  gives  a  normal 
result,  that  is  it  furnishes  the  same  quantity  of  sugar  as  if  it 
had  not  been  heated.  In  3  per  cent  starch  the  infusion,  kept 
at  68°,  furnishes  a  still  larger  amount  of  sugar. 

The  variation  in  the  energy  of  the  infusion,  according  to 
the  concentration  of  the  starch,  becomes  more  striking  if  one 
observes  that  in  the  three  experiments  cited  above  there  are 
formed  quantities  of  maltose  proportional  to  the  amounts  of 
starch  contained  in  the  paste.  There  is  always  obtained  30 
per  cent  of  maltose. 

The  heated  infusion  then  preserves  all  its  properties 
when  it  is  a  question  of  producing  limited  decompositions,  as 
long  as  hydration  does  not  exceed  30  per  cent,  but  this  in- 
fusion cannot  produce  greater  hydration. 

Workers  have  sought  to  explain  the  difference  of  action 
of  the  heated  diastase  and  that  which  is  not  heated  by  the 
hypothesis  that  there  exist  different  kinds  of  amylase.  These 
different  diastases  would  possess  different  temperatures  of 
destruction  and  coagulation  and  would  decompose  starch 
differently.  According  to  this  theory,  by  heating  the  infusion 
to  68°,  an  unfavorable  action  would  be  exerted  on  the  dia- 

es  producing  complete  hydration,  that  is,  giving  little 
dextrin  and  much  sugar,  but  the  diastases  performing  the 
opposite  work,  that  is,  forming  much  dextrin  and  little  sugar, 
would  be  left  intact. 

The  diastases  which  produce  a  slight  saccharification 
would,  according  to  this  hypothesis,  act  very  favorably  when 


H2  THE  ENZYMES  AND    THEIR   APPLICATIONS 

put  in  a  heated  infusion  of  large  quantities  of  starch.  In  this 
particular  case  it  would  be  found  that  the  height  of  the 
temperature  produced  no  change  in  the  diastases. 

We  shall  have  occasion  to  return  to  this  hypothesis,  but 
let  us  say,  at  this  point,  that  it  does  not  at  all  accord  with  the 
facts  which  we  shall  set  forth  later. 

Influence  of  Chemical  Agents. — The  conditions  of  the 
medium  influence  to  a  very  great  degree  the  action  of  amy- 
lase, which  shows  itself  very  sensitive  towards  many  chemi- 
cal substances. 

For  a  long  time  it  has  been  observed  that  the  least 
change  in  the  reaction  of  the  medium  has  a  visible  influence 
on  the  progress  of  saccharification  by  the  diastase.  It  is 
generally  admitted  that  diastatic  action  is  favored  by  very 
slight  amounts  of  acid  and  that,  by  a  greater  acidity,  it  is 
possible  to  slacken  and  then  arrest  completely  the  progress, 
of  hydration. 

Kjeldahl  was  the  first  to  study  with  exactness  the  influ- 
ence of  the  acidity  of  the  medium.  For  this  experiment  he 
made  use  of  dextrin  solutions.  In  a  series  of  samples  of  ioo 
cubic  centimetres  he  added,  to  the  same  quantities  of  in- 
fusion of  malt  and  of  starch,  different  quantities  of  sulphuric 
acid,  allowed  the  saccharification  to  continue  for  20  minutes 
at  a  temperature  of  590,  and  then  determined  the  sugar 
formed.  The  influence  of  the  different  amounts  of  acid  upon 
the  diastase  is  shown  in  the  following  table : 

Milligrams  of  H2S04  Increase  in 

per  100  c.c.  of  solution.  sugar. 

0      0.44 

1 O.47 

2         O.49 

2-5     , O.48 

3  0-43 

3-5  o-27 

4  0.13 

6  0.02 

10 0.01 


AMYLASE.  113 

Thus  it  is  seen  that  an  amount  of  1  to  2.5  milligrams  of 
sulphuric  acid  produces  a  favorable  action,  while  an  amount 
of  3.5  milligrams  causes  a  slackening  which  larger  amounts 
accentuate  more  and  more.  With  10  milligrams  there  is  ob- 
tained an  almost  complete  arrest. 

If  one  compares  sucrase  and  amylase  from  the  point  of 
view  of  their  sensitiveness  towards  the  reaction  of  the 
medium,  one  finds  a  very  noticeable  difference  between  the 
two  enzymes. 

We  have  seen  that  sucrase  produces  its  maximum  effect 
in  an  acid  medium.  On  the  other  hand,  the  favorable  influ- 
ence exerted  by  acids  is  extremely  slight  for  amylase. 

For  the  diastase  which  inverts  cane-sugar  the  natural 
medium  is  acid.  On  the  contrary  the  enzyme  producing 
maltose  derives  little  benefit  from  a  slight  acidity,  and  shows 
an  extraordinary  sensitiveness  towards  larger  quantities  of" 
acid. 

The  figures  indicated  by  Kjeldahl  must  not,  however,  be 
considered  as  constant,  for,  under  conditions  other  than  his, 
we  have  found  entirely  different  results.  We  have  taken  an 
infusion  of  filtered  malt  and  have  added  to  it  different  quanti- 
ties of  sulphuric  acid  and  hydrochloric  acid,  then  we  have  de- 
termined the  diastatic  power  of  the  infusion  before  and  after 
acidification.     We  have  thus  obtained  the  following  results: 

...  Milligrams  per  Diastatic 

100  c.c.  power. 

O     IOO 

2     I08 

Sulphuric  acid J     3    104 

5    100 

10    98 

f    3    107 

Hydrochloric  acid  .J     5    104 

[  IO  97 

Now,  with  10  milligrams  of  sulphuric  acid,  Kjeldahl  found 
an  almost  complete  arrest  in  the  saccharification,  while  in 


H4  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

our  experiments  this  amount  of  acid  showed  itself  almost 
without  effect.  One  may  then  conclude  that  the  medium  by 
itself  possesses  here  also'  an  influence  upon  the  sensitiveness 
of  the  enzyme.  When  a  mineral  acid  is  added  to  an  infusion 
of  malt,  a  part  of  this  acid  combines  with  the  bases  of  the 
infusion,  thus  displacing  organic  acids  which,  varying  in  na- 
ture in  different  infusions,  act  more  or  less  energetically  on 
amylase. 

The  action  of  lactic  acid  on  amylase  deserves  special  at- 
tention because  the  infusion  of  malt,  as  well  as  the  grain 
mashes  generally,  contain  this  acid.  It  is  proper  then,  in  in- 
dustrial saccharification,  to  take  account  of  these  factors.  We 
have  studied  the  action  of  lactic  acid  under  very  varied  con- 
ditions, and  these  experiments  have  led  us  to  the  following 
conclusions :  The  effect  of  a  given  amount  of  acid  on  dia- 
stase differs  according  to  the  duration  of  the  action  and  also 
according  to  the  temperature.  Acids,  furthermore,  act  dif- 
ferently upon  the  saccharifying  power  and  the  liquefying- 
power. 

The  combined  influences  of  time  and  acids  may  be  shown 
by  the  following  experiment:  To  an  infusion  of  malt,  fil- 
tered by.  the  Chamberland  filter,  add  different  quantities  of 
lactic  acid,  and  determine  the  diastatic  power  after  i  hour 
and  after  12  hours. 

Saccharifying  power  of  the  infusion. 
Lactic  acid  per  ioo  c.c.  J     ° 

Centigrams.                        ^~fter  x  faoun                   After  M  hours_ 
IO    48     . 42 

100 53 24 

400   57   21 

By  leaving  the  infusion  of  malt  for  an  hour  with  400  cen- 
tigrams of  acid  at  a  temperature  of  300,  a  perceptible  increase 
of  the  saccharifying  power  is  observed,  while  the  same 
amount  of  acid  produces  a  disastrous  effect  if  the  action  is  al- 
lowed to  be  prolonged  for  12  hours.  The  saccharifying 
power  falls  off  in  this  case  from  57  to  21. 


AMYLASE.  115 

Then  the  same  experiment  is  repeated,  only  changing  the 
temperature.  The  infusion  is  left  at  a  temperature  of  550 
for  1  hour  and  the  diastatic  power  determined. 

Acid  in  centigrams.  Diastatic  power. 

10    44 

100   41 

400   20 

The  amounts  of  acid  which  produced  an  increase  of  cMastatic 
power  at  300  act  quite  otherwise  at  a  temperature  of  55 °. 
At  this  temperature  the  400  centigrams  of  acid  have  made 
the  saccharifying  power  fall  off  from  40  to  20. 

The  sensitiveness  of  amylase  becomes  still  more  evident 
if  we  examine  the  changes  produced  in  the  liquefying  power, 
after  1  and  12  hours  of  action,  with  different  quantities  of 
acid. 

T        .         .  ,  Liquefying  power. 

Lactic  acid  *  >.        

in  centigrams.                      After  I  hour.                     After  12  hours. 
IO IOO    IOO 

100 100   50 

400 51     20 

The  changes  observed  in  the  liquefying  power  show  us 
that  under  the  influence  of  acids  the  increase  in  the  sacchari- 
fying power  of  amylase  is  made  with  a  parallel  reduction  of 
the  liquefying  power. 

After  1  hour  at  300,  400  centigrams  cause  the  saccharify- 
ing power  to  rise  from  48  to  57,  but  at  the  same  time  the 
liquefying  power  is  found  to  be  reduced  almost  one-half. 

We  shall  see  further  on,  in  the  chapter  devoted  to  indus- 
trial applications,  that  the  real  value  of  the  diastase  exists  in 
its  liquefying  power  and  that  consequently  the  rise  caused  by 
acid  is  more  apparent  than  real. 

Alkaline  media  are  unfavorable  to  the  action  of  amylase. 
Still,  the  diastase  can  endure  a  certain  alkalinity  without 


n6  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

changing,  for,  when  the  alkali  is  neutralized,  the  diastase 
resumes  its  activity. 

Sodium  carbonate  acts  upon  amylase  in  extremely  small 
amounts.  By  adding  5  milligrams  of  sodium  carbonate  to 
100  cubic  centimetres  of  neutral  starch,  we  have  found  the 
diastatic  power  to  diminish  almost  20  per  cent.  With  .25 
gr.  of  soda  we  have  obtained  only  a  quarter  of  the  quantity 
of  sugar  which  the  diastase  would  have  given  without  the 
addition  of  alkali.  According  to  the  experiment  of  Duggan, 
caustic  soda  in  the  amount  of  2  milligrams  produces  a  disas- 
trous effect :  under  its  influence  amylase  loses  almost  75  per 
cent  of  its  activity. 

Salts  also  influence  diastatic  action,  by  either  increasing 
or  decreasing  the  activity  of  the  amylase. 

Mercuric  chloride  in  the  amount  of  one-millionth 
paralyzes  its  action. 

Calcium  chloride  in  the  amount  of  one-hundredth  dimin- 
ishes the  activity  of  amylase  by  half. 

According  to  Kjeldahl,  the  salts  of  lead,  zinc,  and  iron, 
as  well  as  alum,  check  the  action  of  the  diastase  and  their 
more  or  less  destructive  influence  may  be  expressed  by  the 
following  figures  which  indicate  the  proportion  between  the 
normal  action,  expressed  by  100,  and  the  action  with  salts: 

Potassium  nitrate,  10  centigrams 20 

Zinc  sulphate 20 

Ferrous  sulphate 20 

Alum   3 

According  to  different  authors,  sodium  chloride  in  the 
amount  of  one-half  per  cent  would  cause  a  noticeable  slack- 
ening. Our  experiments  have  not  confirmed  these  data. 
With  commercial  salt  we  have  often  observed  a  paralyzing  ac- 
tion, but  the  same  thing  has  not  occurred  when  we  have  used 
chemically  pure  salt.  The  checking  action  of  commercial 
common  salt  must  then  be  attributed  rather  to  impurities. 


AMYLASE.  117 

Alcohol  and  most  antiseptics  must  also  be  placed  in  the 
class  of  inhibiting  agents.  Salicylic  acid,  phenol,  and  formic 
aldehyde,  used  in  the  smallest  amounts,  act  upon  the  dia- 
stase. 

Still  we  cannot  definitely  place  all  antiseptics  in  the  class 
of  destructive  substances.  Picric  acid,  for  example,  as  is 
shown  by  our  experiments,  does  not  act  at  all  as  a  depressor ; 
its  action  is  shown  rather  in  the  opposite  direction. 

The  study  of  the  conditions  which  may  influence  the  prog- 
ress of  saccharification  presents  a  genuine  interest  from  a 
practical  point  of  view.  It  furnishes  valuable  information  to 
distillers  and  brewers  as  well  as  other  manufacturers  who 
utilize  the  properties  of  amylase. 

In  seeking  to  determine  conditions  favoring  diastatic  ac- 
tion, we  have  attempted  to  effect  the  saccharification  by  the 
aid  of  a  great  number  of  chemical  substances.  These  experi- 
ments have  not  fulfilled  all  our  hopes.  We  have  not  suc- 
ceeded in  enhancing  the  efficiency  of  a  malt  by  chemical  sub- 
stances, but  the  results  of  our  experiments  throw  a  special 
light  on  the  mode  of  action  of  amylase  and  afford  a  firm  basis 
for  analysis  of  the  diastase. 

We  have  found  that  many  chemical  substances  may  re- 
inforce to  a  very  great  degree  the  progress  of  saccharifica- 
tion by  the  diastase.  This  reinforcing  action  is,  however,  of 
a  peculiar  nature,  and  can  only  be  shown  under  certain  con- 
ditions. To  the  list  of  favorable  substances  belong  the  salts 
of  vanadium  and  aluminium,  the  phosphates,  asparagin,  albu- 
minoid substances,  and  picric  acid.  To  study  the  action  of 
these  different  substances  on  diastatic  fermentation  we  have 
used  two  different  methods. 

The  diastase  has  first  been  put  in  direct  contact  with  the 
reagent  and  then  introduced  into  the  starch.  After  sac- 
charification the  quantity  of  sugar  formed  has  been  meas- 
ured. In  a  parallel  experiment,  made  with  the  same  quan- 
tity of  diastase  not  having  undergone  the  influence  of  the 
reagent,  the  quantity  of  maltose  produced  has  been  deter- 


n«  THE   ENZYMES  AND    THEIR   APPLICATIONS. 

mined  and  finally  the  results  of  the  two  experiments  have 
been  compared.  In  the  second  series  of  experiments  the  re- 
agents have  been  added  directly  to  the  starch,  in  which  is 
then  poured  the  infusion  of  malt. 

In  our  experiments  we  used  an  infusion  of  malt  prepared 
cold  with  one  part  of  malt  and  forty  parts  of  water.  The 
starch  solution  had  a  density  of  1.015.  For  each  experiment 
we  used  1  cubic  centimetre  of  filtered  infusion  and  100  cubic 
centimetres  of  starch.  The  saccharification  took  place  at 
500  for  1  hour. 

Here  are  some  figures  which  sum  up  the  influence  of  the 
different  chemical  substances : 

Maltose  per  ioo 
of  starch. 

Without  addition 8.63 

With    0.7  gr.   of  ammonium  phosphate..    51.62 

0.5       acid  calcium  phosphate 46.12 

0.25     aluminium  acetate 62.40 

0.25     ammonium  alum 56.30 

0.25     potassium  alum 54-32 

0.05     asparagin 61.20 

By  the  addition  of  50  milligrams  of  asparagin  the  sac- 
charification was  nearly  7  times  more  extensive  than  in  the 
proof  experiments. 

Aluminium  acetate  can  produce  the  same  effect,  but  it 
must  be  used  in  a  larger  quantity. 

The  two  methods  which  we  have  used  have  afforded  dif- 
ferent results  for  calcium  phosphate  as  well  as  for  alum. 

For  the  other  substances  we  could  find  no  difference. 

The  results  are  not  changed  if  instead  of  an  infusion  of 
malt,  diastase  precipitated  by  alcohol  is  used.  Neither  do 
they  change  if  the  saccharification  is  effected  at  different 
temperatures.  It  is  very  evident  that  the  quantities  of  sugar 
found  vary  according  to  the  temperature  of  saccharification, 
Imt  the  difference  between  the  proof  experiments  and  the 


AMYLASE.  119 

experiments  made  with  various  substances  always  remains 
practically  the  same. 

A  series  of  experiments  made  under  widely  differing  con- 
ditions has  led  us  to  the  following  conclusions: 

1st.  Substances  which  act  favorably,  act  in  proportion  to 
their  quantity  up  to  a  certain  maximum  amount. 

Thus,  by  taking  0.005  of  asparagin,  we  have  obtained  25.5  maltose. 
0.02  "  "  37 

"  0.05  "  "  61.2         " 

"  1  gr.  "  "  61.2         " 

2nd.  The  maximum  is  not  the  same  for  all  substances 
favoring  diastatic  action. 

Thus,  asparagin  and  aluminium  acetate  in  maximum 
quantities  may  influence  diastatic  fermentation  much  more 
strongly  than  phosphates. 

3rd.  The  exciting  action  of  chemical  substances  is  mani- 
fested only  in  the  first  phase  of  hydration  of  starch ;  when 
saccharification  is  very  advanced  it  ceases  to  act. 

It  results  from  these  facts  that  the  same  substance  may 
possess  a  very  different  activity  according  to  the  conditions 
of  the  experiment. 

If  amylase  is  present  in  a  very  small  proportion  in  rela- 
tion to  the  starch  to  be  transformed,  the  effect  of  chemical 
substances  is  easy  to  ascertain.  In  the  opposite  case,  that  is 
with  a  greater  quantity  of  amylase,  the  effect  of  chemical 
substances  is  reduced,  and,  for  a  quantity  of  diastase  capable 
of  itself  transforming  nearly  60  per  cent  of  starch,  the  ac- 
celerating substances  have  no  longer  any  influence  on  the 
enzyme. 

The  following  experiment  shows  the  influence  of  aspar- 
agin with  different  quantities  of  infusion. 

In  each  of  two  portions  of  100  cubic  centimetres  of  starch 
paste  is  introduced  1  cubic  centimetre  of  malt  infusion  and 
saccharification  is  allowed  to  proceed  for  an  hour  at  500. 
One  of  the  specimens  is  saccharified  without  addition  of  as- 
paragin; to  the  other  is  added,  when  the  enzyme  is  intro- 
duced, 5  centigrams  of  that  substance. 


120  THE  ENZYMES  AND   THEIR  APPLICATIONS 

At  the  end  of  the  time  interval,  one  determines  the  pro- 
portion of  maltose  formed  ia  the  solution  of  starch  submitted 
to  diastatic  action.  These  same  experiments  are  then  re- 
peated under* the  same  conditions  with  10  cubic  centimetres 
of  infusion  in  place  of  one,  and  the  followiing  results  are  ob- 
tained : 

Maltose  per  ioo. 

j     i   c.c.  without  asparagin 18 

"     with  asparagin 62 

10  c.c.  without  asparagin 79-25 


with  asparagin 79-25 

It  appears  from  this  table  that  in  the  experiment  contain- 
ing 10  cubic  centimetres  of  infusion,  the  asparagin  no  longer 
acts,  although  the  saccharification  must  still  be  far  from 
finished. 

An  analogous  result  may  be  obtained,  even  with  a  very 
small  quantity  of  diastase.  For  this,  instead  of  checking  sac- 
charification after  an  hour,  as  was  just  done,  the  diastase  is 
left  in  contact  with  the  starch  for  twelve  hours. 

Per  cent  of  maltose. 

f  Saccharification,  1  hour  at  300. 

A  -{    1)   1   c.c.  of  infusion  without  asparagin.  .  6.4 

t  2)                          "       with  asparagin 45,0 

f  Saccharification,  12  hours  at  300. 

B  -^    1)   1   c.c.  without  asparagin 74.8 

I  2)          "    with  asparagin 74.9 

Another  characteristic  property  of  substances  favoring 
diastatic  fermentation  is  that  they  act  exclusively  on  the  sac- 
charifying power,  while  they  never  influence  the  liquefying 
power  of  amylase. 

As  the  liquefying  power  exerts  its  action  exclusively  on 
starch  and  not  on  dextrins,  it  must  be  assumed  that  the  ac- 
tion of  favoring  substances  is  exerted  only  on  the  latter 
bodies. 


CHAPTER  IX. 

CHEMICAL  WORK  OF  AMYLASE. 

Chemical  work  of  amylase. — Theories  of  Payen  and  Musculus. — Existence 
of  different  dextrins. — Theory  of  Duclaux  on  the  nature  of  the  dif- 
ferent dextrins. — Preservation  of  the  diastases  during  saccharification. 
— Experiments  of  Effront. 

When  grains  of  starch  are  submitted  for  a  short  time  at 
a  low  temperature  to  the  action  of  amylase,  they  are  very 
slightly  attacked  by  the  diastase.  On  the  contrary,  if  the 
action  is  prolonged,  a  very  complete  work  is  effected;  the 
grains  are  corroded,  pass  into  solution  and  are  then  trans- 
formed into  sugars  and  related  bodies.  The  action  of  amy- 
lase is  nevertheless  more  energetic  and  much  more  rapid 
when  it  is  produced  on  starch  paste.  By  allowing  amylase 
to  act  at  a  suitable  temperature  on  starch  paste  the  solution 
and  saccharification  proceed  rapidly.  The  chemical  reactions 
that  the  diastase  cause  in  the  paste,  as  well  as  on  the  grains 
of  starch,  may  be  expressed  by  the  following  equation : 

(C12H20Oi0)  +  H20  =  C12H22011. 

Starch.  Maltose. 

This  formula  shows  us  that  starch,  under  the  influence  of 
amylase,  is  hydrated  and  transformed  into  maltose,  but  it 
does  not  indicate  the  mechanism  of  the  transformation.  In 
reality  the  phenomenon  is  much  more  complex. 

In  the  products  of  the  reaction  there  are  always  found 
dextrins  whose  presence  shows  that  the  reaction  was  com- 
plicated by  the  formation  of  intermediate  products. 


122  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

The  saccharification  of  starch  by  malt  has  been  the  object, 
of  much  research.  However,  in  spite  of  the  number  of  re- 
searches in  this  field,  the  complete  solution  of  the  problem  is 
far  from  being  found  at  present. 

The  simplest  and  also  the  oldest  interpretation  of  the 
course  of  saccharification  is  that  of  Payen.  According  to 
him,  diastase  exercises  on  starch  two  successive  actions :  it 
first  transforms  it  into  dextrin,  then  into  maltose.  There  is 
first  produced  an  isomeric  modification  of  starch,  then  a  hy- 
dration of  that  isomer.  According  to  the  interpretation  of 
Payen  the  transformation  of  starch  into  dextrin  and  into 
maltose  would  of  necessity  be  produced  not  only  gradually 
but  regularly,  from  the  beginning  to  the  end  of  the  action. 
Now,  the  course  of  saccharification  presents  quite  a  different 
aspect.  We  know,  in  fact,  that  as  hydration  proceeds  the 
action  of  amylase  becomes  slower  and  slower.  The  theory 
of  Payen  is  then  in  disagreement  with  the  facts.  Further- 
more, it  cannot  explain  to  us  the  formation  during  sacchari- 
fication of  dextrins  endowed  with  different  properties. 

According  to  Musculus,  saccharification  takes  place,  not 
by  the  successive  transformation  of  starch  into  dextrin  and 
then  into  maltose,  but  by  hydration  followed  by  cleavage. 
This  author  maintains  that  the  molecule  of  starch  is  first 
hydrated  and  then  decomposed  into  a  molecule  of  maltose 
and  a  molecule  of  dextrin. 

2C12-ti20O10    T"    xi20    " —    ^12^22^11    "T    ^12^20^'l0* 


Starch.  Maltose.  Dextrin. 

In  support  of  this  theory  Musculus  endeavors  to  establish 
that  the  dextrin  and  sugar  formed  during  saccharification 
are  in  a  constant  ratio.  He  insists,  moreover,  that  dextrin 
cannot  be  attacked  by  the  diastase.  These  facts  do  not  with- 
stand criticism.  In  fact,  dextrins  can  be  transformed  into 
sugar  by  amylase,  and  the  ratio  between  the  quantities  of 
maltose  and  of  dextrins  does  not  remain  at  all  constant  dur- 


CHEMICAL    WORK  OF  AMYLASE.  1 23 

ing  the  transformation.  This  ratio  changes  for  the  same 
temperature  according  to  the  duration  of  the  action  and  the 
quantity  of  diastase  used,  and  also  depends  upon  the  tem- 
perature of  saccharification.  The  ratio  between  the  prod- 
ucts formed,  the  maltose  and  the  dextrins,  is  then  neither 
simple  nor  constant. 

The  theory  of  Musculus,  based  upon  observations  which 
are  not  very  exact  and  reasonings  which  are  not  welt 
founded,  has  however  had  unmerited  acceptance,  and  at  the 
present  time  it  still  serves  as  a  basis  for  almost  all  the 
theories  of  saccharification.  To  reconcile  this  theory  with 
the  data  on  saccharification  actually  possessed,  a  repetition 
of  the  two  operations,  hydration  and  cleavage,  is  believed  to 
occur.  The  starch  is  supposed  to  possess  a  very  great  molec- 
ular weight.  This  complex  molecule,  by  hydrating,  is  de- 
composed into  maltose  and  a  primary  dextrin.  This  dextrin, 
of  a  complicated  constitution,  then  furnishes  in  its  turn  a 
second  molecule  of  maltose  and  a  new  dextrin  of  a  molecular 
weight  less  than  that  of  the  first,  and  so  on. 

The  saccharification  is  then  brought  about  by  giving  rise 
successively  to  dextrins  of  smaller  and  smaller  molecular 
weight. 

The  ideas  of  Musculus  as  to  the  course  of  saccharification. 
explaining  it  as  a  progressive  degradation  of  the  dextrins, 
were  adopted  by  Brown  and  Morris  as  well  as  by  Lihtner. 
They  are,  however,  far  from  sharing  the  opinion  of  Musculus 
as  to  the  formation  of  the  intermediate  products  of  the  re- 
action, and  as  to  the  molecular  weight  of  the  starch  and  the 
dextrins. 

According  to  other  chemists,  among  the  products  of  sac- 
charification there  are  found  not  only  dextrins  and  maltose, 
but  also  substances  formed  by  the  combination  of  these  two 
bodies. 

Existence  of  Different  Dextrins. — It  does  not  enter  into 
the  scope  of  the  present  work  to  discuss  all  the  theories 
evolved  to  explain  the  saccharification  of  starch.     Let  us 


324  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

confine  ourselves  to  the  essential  facts  underlying  these 
theories,  especially  the  formation,  during  sacchanfication,  of 
dextrins  which  act  differently  towards  reagents. 

To  show  the  difference  between  the  dextrins,  one  may 
proceed  in  the  following  manner : 

Treat  with  alcohol  a  paste  of  saccharified  starch  contain- 
ing from  10  to  20  parts  of  sugar  for  100  parts  of  starch.  Dis- 
solve the  precipitate  obtained,  precipitate  again  with  alcohol 
and  repeat  this  operation  a  number  of  times.  Thus  a  prod- 
uct is  secured  which  contains  only  traces  of  sugar. 

On  the  other  hand,  precipitate  in  the  same  manner  the 
dextrins  contained  in  a  paste  of  starch  in  an  advanced  stage 
of  saccharification  and  containing  nearly  80  parts  of  maltose 
ior  100  parts  of  starch.  With  the  two  kinds  of  dextrins  thus 
obtained  and  which  we  will  call  dextrins  A  and  B,  prepare 
two  solutions  of  the  same  concentration ;  add  to  them  the 
same  quantity  of  infusion  of  malt  and  allow  saccharification 
to  proceed  at  500  for  2  hours. 

A  measurement  of  the  maltose  in  the  two  solutions  shows 
the  difference  existing  between  the  two  dextrins. 

The  dextrin  A,  extracted  from  slightly  saccharified  starch, 
5s  hydrated  very  easily,  while  dextrin  B  furnishes  very  little 
maltose.  The  two  dextrins  then  differ  in  their  sensitiveness 
towards  amylase. 

The  difference  in  the  nature  of  the  two  dextrins  can 
he  still  further  shown  by  the  action  of  acids.  Large  amounts 
•of  a  mineral  acid,  acting  while  warm  on  the  two  dextrins, 
give  with  both  equal  quantities  of  dextrose,  but  different 
results  are  obtained  if  very  slight  amounts  of  acid  are  used. 
In  this  case,  very  marked  differences  are  observed  in  the 
progress  of  hydration:  the  dextrins  coming  from  slightly 
saccharified  mashes  are  transformed  under  the  influence  of 
acids,  much  more  easily  than  others. 

Another  difference  between  the  two  dextrins  is  shown  by 
the  clearly  different  action  exerted  upon  them  by  diastases 
with  accelerating  substances.     Take  two  specimens  of  the 


CHEMICAL    WORK  OF  AMYLASE.  125 

solution  of  dextrin  A,  to  which  add  equal  quantities  of  malt 
infusion  and  to  one  add  also  a  slight  amount  of  asparagin. 

By  letting  the  diastase  act  for  some  time  and  then 
measuring  the  sugar  formed,  it  is  found  that  the  saccharifica- 
tion  is  produced  in  a  very  different  manner  in  the  two  cases. 

The  specimen  containing  the  asparagin  shows  itself  much 
richer  in  sugar  than  the  specimen  lacking  the  accelerating 
substance. 

If  the  same  experiments  are  now  repeated  with  the  dex- 
trin B,  it  is  ascertained  that  the  progress  of  saccharification 
is  entirely  different.  The  two  experiments,  that  without 
asparagin  as  well  as  that  which  contains  it,  have,  after  sac- 
charification, the  same  quantity  of  sugar.  This  proves  that 
dextrin  B  is  not  susceptible  to  the  combined  actions  of  amy- 
lase and  asparagin,  while  the  transformation  of  dextrin  A  is 
influenced  by  the  united  action  of  these  two  substances. 

The  difference  in  sensitiveness  of  the  dextrins  A  and  B 
explains  to  us  the  irregularity  in  the  progress  of  saccharifica- 
tion. It  gives  us  at  the  same  time  the  reason  of  the  lack  of 
ratio  between  the  quantities  of  diastase  used  and  of  maltose 
formed. 

It  is  the  formation,  at  the  end  of  the  reaction,  of  dextrins 
of  a  special  nature  which  produces  the  slackening  in  the 
progress  of  hydration  and  which  destroys  the  proportionality 
between  the  quantities  of  active  substance  and  product 
formed,  which  exists  at  the  beginning  of  hydration  before 
the  final  dextrins  are  formed. 

The  existence  of  different  dextrins  is  an  argument  in 
favor  of  the  theory  which  regards  saccharification  as  hydra- 
tion followed  by  cleavage.  The  authors  of  this  hypothesis 
are,  however,  wrong  in  bringing  forward,  to  demonstrate  the 
existence  of  different  dextrins,  arguments  of  little  value,  and 
to  attribute  to  these  different  dextrins  properties  which  they 
do  not  have. 

Thus,  according  to  many  authors,  dextrins  would  be  dis- 
tinguished from  each  other  by  the  difference  in  their  rotatory 


126  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

and  reducing  powers.  Now,  in  reality,  these  differences  do 
not  exist  at  all.  The  differences  found  between  the  reduc- 
ing powers  and  the  rotatory  powers  of  different  dextrins 
come  solely  from  the  impurities  which  these  dextrins  con- 
tain. They  arise  specially  from  the  admixture  of  sugar  which 
is  removed  with  difficulty,  even  by  repeated  precipitations 
with  alcohol. 

To  obtain  dextrins  free  from  sugar,  we  submit  impure 
dextrins  to  an  alcoholic  fermentation  as  wTell  as  to  lactic  fer- 
mentation. The  different  dextrins  obtained  by  this  method 
possess  neither  rotatory  nor  reducing  power.  Moreover, 
the  characteristic  properties  of  the  dextrins  A  and  B  prove 
sufficiently  the  existence  of  different  bodies  of  this  class  and 
is  is  not  at  all  necessary  to  attribute  to  them  still  other  char- 
acteristics which  they  do  not  possess. 

The  theory  of  Musculus  in  its  modern  form  assumes  that 
the  dextrins  differ  in  their  molecular  weight.  By  employing 
Raoult's  method  of  freezing  it  has  not  been  possible  to  es- 
tablish with  certainty  that  dextrins  obtained  after  more  or 
less  extensive  saccharification  really  possess  different  molec- 
ular weight.  Lintner  and  Dull  have  found  for  erythro- 
dextrin  a  molecular  weight  of  6000,  and  for  the  other  dex- 
trins a  molecular  weight  of  2000.  These  figures  must,  how- 
ever, be  taken  with  a  certain  reserve,  for  Lintner  and  Dull 
ascertained  at  the  same  time,  for  various  dextrins,  the  exist- 
ence of  rotatory  and  reducing  powers.  It  is,  therefore,  to  be 
presumed  that  their  determinations  were  made  with  impure 
products,  and  that  under  those  conditions  they  have  only  a 
relative  value. 

Theory  of  Duclaux  on  the  Origin  of  the  Different  Dex- 
trins.— The  existence  of  different  dextrins  being  demon- 
strated, one  may  ask  from  whence  they  arise  and  by  what 
mechanism  they  are  produced  during  the  course  of  sacchari- 
fication. 

According  to  Duclaux  it  is  in  the  structure  of  the  mole- 
cules of  starch  that  one  must  look  for  the  origin  of  the  dif- 


CHEMICAL   WORK  OF  AMYLASE. 


127 


ferences  found  between  the  various  products  of  its  trans- 
formation. 

According  to  this  author  dextnns  differ  from  each  other, 
not  by  their  chemical  structure  but  by  their  physical  consti- 
tution. These  differences  have  for  cause  the  structure  of 
the  grains  of  starch,  which  are  composed  of  non-homoge- 
neous superposed  layers,  unevenly  compact,  and  offering  a 
different  resistance  to  physical  and  chemical  agents.  This 
hypothesis,  very  attractive  because  of  its  simplicity,  is  sup- 
ported by  very  weighty  arguments.  It  has  been  known  for 
a  long  time  that  starch  acts  differently,  according  to  its 
origin,  in  the  presence  of  amylase  in  the  cold.  Potato-starch 
is  very  difficultly  attacked,  while  the  starches  of  barley  and 
wheat  saccharify  with  great  ease. 

This  difference,  which  evidently  comes  from  the  more  or 
less  compact  state  of  the  layers  forming  the  grain  of  starch, 
is  again  met  with  in  the  action  of  this  diastase  at  relatively 
high  temperatures. 

Lintner,  by  saccharifying  raw  starches  of  different  ori- 
gins, has  found  that  the  attack  varies  considerably  in 
energy  according  to  the  origin  of  the  starch. 

The  proportions  of  starch  dissolved  at  different  tempera- 
tures are  here  tabulated : 


Potato  

Barley 

Fresh  malt , 
Brewed  "    . 

Wheat 

Rice 

Maize 

Rye 


Temperature  of  Action. 


O 
12 
29 
13 


6 
2 

25 


5 
53 
58 
56 
62 

9 


52 
92 
92 
91 
91 
19 
18 
40 


90 
96 
96 
93 
94 
3i 
54 
94 


Temperature 

of 
Gelatimzation 


65 

80 


75-80 
80 

75 


At  a  temperature  of  500,  12  per  cent  of  barley-starch  is 
dissolved,  2  per  cent  of  corn-starch,  and  25  per  cent  of  rye- 
starch.  At  a  temperature  of  6o°,  92  per  cent  of  barley- 
starch  is  dissolved  and  only  18  per  cent  of  corn-starch.     The 


128  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

quantity  of  starch  which  can  be  dissolved  at  a  given  tem- 
perature, "therefore,  depends  distinctly  upon  the  origin  of  the 
starch.  Noticeable  differences  are  also  found  between  the 
temperatures  necessary  for  the  gelatinization  of  starches  of 
different  origin.  Potato-starch  gelatinizes  at  65  °,  and  the 
diastase  at  this  temperature  dissolves  90  per  cent,  while  the 
barley-starch,  which  gelatinizes  at  much  higher  tempera- 
tures, yields  at  650,  96  per  cent  of  dissolved  substances. 

These  figures  prove  that  gelatinization  does  not  change 
the  properties  of  starch  which  result  from  the  variable  de- 
gree of  compactness  of  the  different  layers  of  grains. 

Really,  a  granule  of  starch  is  irregularly  attacked  by  dia-. 
stase :  corrosion  occurs  in  very  different  directions  and 
places.  This  manner  of  corrosion  arises  from  the  inequality 
of  resistance  of  the  surface  of  the  grains,  so  that  the  differ- 
ence existing  in  the  compactness  of  the  various  parts  of  the 
grains  is,  on  the  whole,  the  initial  cause  of  the  variations  in 
resistance  to  diastatic  action. 

Potato-starch  and  barley-starch  are  both  composed  of 
non-homogeneous  granules  differing  in  the  degree  of  com- 
pactness of  the  layers  which  compose  them. 

In  the  granules  of  potato-starch  more  resisting  layers  are 
found  than  in  the  granules  of  barley-starch.  Now,  we  have 
observed  that  with  different  kinds  of  starch,  pastes  are  ob- 
tained which  saccharify  with  more  or  less  difficulty.  We 
must  then  suppose  that  the  difference  of  compactness  be- 
tween parts  of  the  same  granule  does  not  disappear  when  the 
starch  gelatinizes  and  that  consequently  the  starch  cannot 
offer  an  equal  resistance  in  all  its  parts. 

The  most  coherent  parts  of  the  granules  will  form  a  paste 
more  difficult  to  liquefy  and  will  then  give,  even  when  enter- 
ing into  solution,  a  dextrin  which  will  offer  more  resistance. 

From  this  point  of  view,  the  dextrins  do  not  exist  as 
chemically  distinct  bodies,  but  the  different  constituents  of 
the  starch-grains  of  varying  coherence  yield  more  or  less  re- 
fractory dextrins. 


CHEMICAL    IVORK  OF  AMYLASE.  1 29 

According  to  Duclaux,  the  phenomena  occur  in  the  fol- 
lowing order : 

By  the  action  of  amylase  on  the  paste  there  is  first  pro- 
duced an  almost  instantaneous  liquefaction.  There  is  de- 
struction of  a  coagulum,  analogous  to  that  observed  when 
a  few  drops  of  acid  or  of  ammonium  citrate  are  added  to  a 
gelatinous  mass  of  calcium  phosphate.  Saccharification 
then  commences  in  the  least  resistant  portion  of  the  paste. 
This  portion  is  first  transformed  into  dextrin,  then  into  mal- 
tose, but  at  the  same  time  other  portions  of  starch  are  at- 
tacked and  increase  the  quantities  of  dextrin  and  maltose  in 
the  solution. 

When  iodine  no  longer  gives  color,  the  starch  is  com- 
pletely transformed,  but  there  are  still  left  some  dextrins 
arising  from  the  portions  of  starch  which  were  least  easily 
attacked.  Some  of  the  dextrins  are  so  slow  to  disappear 
that  they  still  remain  at  the  end  of  the  operation.  Still,  these 
dextrins  disappear  in  their  turn  and  are  transformed  into 
maltose  if  the  diastatic  action  is  sufficiently  prolonged. 

When  precautions  are  taken  to  avoid  alteration  of  the 
diastase,  the  course  of  saccharification  conforms  to  the 
theory  of  Payen.  The  starch  is  first  transformed  into  dex- 
trin and  then  into  maltose. 

The  hypothesis  of  the  varying  compactness  of  the  con- 
stituents of  the  starch-grains  has  then  brought  a  new  sup- 
port to  Payen's  theory,  which,  at  first  sight,  appeared  to  be 
in  direct  contradiction  to  all  the  data  on  saccharification. 

Deterioration  of  Diastases  by  Work. — In  the  rapid  glance 
which  we  have  just  given  at  the  manner  of  action  of  amylase, 
we  have  been  exclusively  occupied  with  a  single  factor,  the 
progressive  transformation  of  starch,  without  troubling  our- 
selves about  the  fate  of  the  acting  bodies.  The  question 
which  arises  first  of  all  in  considering  the  active  substance 
is  this : 

When  the  diastase  has  carried  out  a  considerable  chem- 


130  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

ical  work,  is  it  still  in  the  same  state  as  at  the  beginning  of 
its  action,  and  has  it  preserved  its  activity? 

This  question  has  been  discussed  by  different  authors. 

According  to  some,  the  diastase  undergoes  a  weakening 
during  the  work;  others  claim  that  it  possesses  at  the  last 
the  same  fermenting  power  as  at  first. 

Unfortunately,  the  two  opinions  are  founded  altogether 
on  very  questionable  general  considerations,  while  the  ex- 
perimental method  alone  can  furnish  the  true  solution  of  this 
question,  so  interesting  from  a  theoretical  point  of  view  and 
so  rich  in  practical  consequences. 

The  following  experiment  gives  the  solution  of  the 
problem: 

In  200  cubic  centimetres  of  starch  paste  put  3  cubic  cen- 
timetres of  a  malt  infusion  and  let  saccharification  proceed 
for  4  hours  at  a  temperature  of  300. 

The  volume  of  liquid  thus  saccharified  is  brought  up  to 
300  cubic  centimetres,  so  that  100  cubic  centimetres  of  the 
liquid  contain  exactly  1  cubic  centimetre  of  infusion  of  malt 
which  has  already  produced  a  work  of  saccharification. 

To  see  if  the  work  done  by  the  infusion  has  really  caused 
weakening  of  the  active  substances,  we  compare  the  fer- 
menting power  of  100  cubic  centimetres  of  this  liquid  with 
the  fermenting  power  of  1  cubic  centimetre  of  the  original 
infusion.  For  this,  100  cubic  centimetres  of  saccharified 
paste  are  mixed  with  200  cubic  centimetres  of  starch.  The 
mixture  is  placed  in  a  water-bath  at  500  for  an  hour.  This 
we  will  call  specimen  A. 

We  also  take  a  second  specimen  of  100  cubic  centimetres 
of  saccharified  paste ;  bring  it  very  rapidly  up  to  a  tempera- 
ture of  ioo°  to  destroy  the  diastase,  after  which  wTe  pour  it 
into  200  cubic  centimetres  of  starch  with  1  cubic  centimetre 
of  fresh  infusion  added  and  place  it  in  the  water-bath.  This 
will  be  specimen  B. 

The  two  saccharifications  are  then  made  during  the  same 
time,  with  the  same  quantities  of  infusion,  but  with  this  dif- 


CHEMICAL    WORK  OF  AMYLASE.  131 

ference,  that  the  infusion  of  specimen  A  has  already  been 
used  once,  while  the  infusion  of  specimen  B  has  not. 

The  quantities  of  maltose  obtained  in  a  series  of  parallel 
experiments  are  here  shown: 

Specimens.       123 

A         1.48   1.31    1.92 

B         1.46   1.32    1.92 

Thus  the  quantity  of  maltose  obtained  is  the  same  in  the 
specimens  A  and  B.  Weakening'does  not,  therefore,  occur, 
and  all  the  theoretical  considerations  from  which  other  con- 
clusions are  drawn  must  be  rejected. 

It  is  true  that  in  changing  the  conditions  of  the  experi- 
ment, exactly  opposite  results  may  easily  be  obtained,  but  in 
such  cases  there  is  alteration  and  not  weakening  of  the  dia- 
stase. 

Thus,  by  repeating  the  same  experiments,  with  the  same 
infusion  and  the  same  starch  and  letting  the  action  be  pro- 
longed, not  4  hours  at  300,  but  only  half  an  hour  at  6o°  or 
65  °,  we  reach  very  different  results. 

Temperature  60°.  Temperature  68°. 

A         2.19  maltose  2.00  maltose 

B         3-^5  "  3-i5         " 

The  differences  found  between  specimens  A  and  B  arise 
here  from  the  action  of  heat  on  the  diastase  and  not  from 
weakening.  The  aqueous  solution  of  amylase,  left  to  a  tem- 
perature of  6o°,  loses  in  fact,  as  is  known,  a  great  part  of  its 
diastatic  power. 


CHAPTER  X. 

AMYLASES  OF  DIFFERENT  SOURCES. 

Different  amylases. — Ptyalin. — Diastase  of  raw  grains  and  diastase  of 
sprouted  grains. — Action  of  translocation  diastase  on  starch. — Reich- 
ler's  diastase. — Mode  of  action  of  diastase  brought  up  to  a  tempera- 
ture of  70°. — Conditions  of  secretion  of  amylase. — Quantitative  analy- 
sis of  amylase. — Comparative  value. — Absolute  value. — Methods  of 
Effront. 

When  we  study  amylases  of  different  sources  from  the 
point  of  view  of  their  action  on  starch,  we  are  struck  with' 
certain  characteristic  peculiarities,  which  tend  to  confirm  the 
existence  of  different  kinds  of  amylase.  The  authors  who 
have  studied  this  question  closely,  first  of  all  distinguish  the 
salivary  diastase  called  ptyalin  and  the  diastase  of  grains. 
They  then  discriminate  between  the  amylase  of  raw  grains 
and  that  of  sprouted  grains. 

The  characteristic  of  the  salivary  diastase  is,  according  to 
certain  authors,  its  resistance  to  the  action  of  alkaline  and 
acid  media. 

This  assertion  is  erroneous.  Ptyalin  really  behaves  to- 
ward the  reactions  of  the  medium  in  exactly  the  same  man- 
ner as  malt  diastase. 

Saliva,  in  reality,  often  possesses  a  very  pronounced  al- 
kaline reaction  which  corresponds  to  97  milligrams  of  bicar- 
bonate of  sodium  per  100  c.c. 

Now,  Chittenden  and  Smith  have  shown  that  this  alkalin- 
ity weakens  the  fermenting  power  of  the  diastase,  and  its 
power  increases  in  a  certain  proportion  when  the  saliva  is 
neutralized. 

132 


AMYLASES   OF  DIFFERENT  SOURCES.  133 

The  resistance  to  acids  which  has  been  claimed  for  ptyalin 
is  especially  based  on  the  part  played  by  the  salivary  diastase 
in  digestion,  but  here  also  the  observations  made  have  not 
been  exact. 

In  fact  ptyalin  acts  only  in  the  first  phase  of  digestion 
■when  the  gastric  contents  are  not  yet  acid.  The  reaction  of 
this  enzyme  is  checked  when  acidity  is  developed. 

The  distinction  between  amylase  of  raw  grains  and  amy- 
lase of  malt  appears  at  first  sight  to  be  based  on  more 
reliable  data.  Lintner  and  Eckhard  have  ascertained  a  per- 
ceptible difference  between  the  action  of  amylase  of  sprouted 
barley  and  that  of  the  diastase  of  barley  which  has  not 
sprouted.  At  low  temperature  the  diastase  of  raw  grains 
accomplishes  a  more  complete  work  than  the  diastase  of 
malt.  At  the  optimum  temperature,  on  the  contrary,  which 
is  practically  the  same  for  the  two  diastases,  it  is  the  amylase 
of  the  sprouted  grain  which  forms  the  greatest  amount  of 
sugar. 

A  still  more  appreciable  difference  results  from  the  study 
of  the  liquefying  power  of  amylases  of  different  origin. 

Malt  diastase  liquefies  starch  very  quickly,  while  the  amy- 
lase of  raw  grains,  though  possessing  an  energetic  sacchari- 
fying power,  shows  itself  almost  inactive  as  regards  liquefac- 
tion. Lintner  and  Eckhard  have  compiled  a  comparative 
table  of  the  action  of  temperatures  on  the  two  diastases  and 
have  brought  forward  many  distinctive  properties,  which, 
they  say,  characterize  the  two  enzymes. 

But  here,  as  in  the  case  of  ptyalin,  the  conclusions  which 
the  experimenters  have  drawn  from  their  observations  are 
not  well  founded. 

The  difference  found  between  an  infusion  of  raw  grain 
and  an  infusion  of  malt  comes  really  not  from  the  existence 
of  two  distinct  diastases,  but  from  the  presence  of  different 
foreign  bodies  in  the  two  liquids.  In  the  infusion  of  raw 
grain  there  is  very  little  amylase,  but  the  liquid  is  very  rich 
in  substances  favoring  diastatic  action. 


134  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

When  we  studied  the  influence  of  substances  which  favor 
diastatic  action,  we  showed  that  this  action  is  manifested 
especially  in  the  first  phase  of  hydration  and  that  it  ceases 
in  the  presence  of  residuary  dextrins.  It  is  for  this  reason 
that  the  low  temperature  is  shown  to  be  favorable  to  the 
action  of  amylase  of  raw  grain  and  unfavorable  to  that  of 
malt  diastase,  which  under  these  conditions  produces  an  in- 
complete hydration. 

At  the  optimum,  temperature  the  conditions  are  entirely 
different.  The  small  quantity  of  diastase  contained  in  raw 
grain  can  by  itself  cause  a  considerable  saccharification 
which  proceeds  up  to  the  point  where  the  foreign  substances 
have  no  more  influence  on  the  course  of  hydration. 

By  using  diastase  of  raw  grain  one  rarely  observes  such 
a  degree  of  saccharification  that  the  iodine  test  for  starch  is 
no  longer  obtained.  The  same  phenomenon  is  observed 
when  the  diastase  is  used  with  accelerating  substances. 

To  obtain  a  decomposition  of  starch  corresponding  to  40 
or  50  per  cent  of  maltose,  a  very  small  amount  of  infusion  is 
sufficient  if  there  is  present  a  body  accelerating  its  action. 
To  reach  70  per  cent  of  maltose  it  needs  10  to  20  times  as 
much  malt  infusion,  even  if  the  work  is  done  in  the  presence 
of  asparagin. 

We  shall  find  later  that  there  are  really  in  raw  grains  sub- 
stances which  accelerate  the  diastase.  These  are  the  cause 
of  all  the  differences  observed  by  Lintner  between  the  dia- 
stase of  malt  and  that  of  grains  which  have  not  sprouted. 

Brown  and  Morris  also  make  a  distinction  between  malt 
amylase  and  the  enzyme  of  raw  grains.  They  call  the  first 
"  secretion  diastase  "  and  the  second  "  translocation  dia- 
stase." According  to  these  authors,  the  two  diastases  act  in 
an  entirely  different  manner  on  raw  starch.  Secretion  dia- 
stase corrodes  the  granules  of  starch,  channels  them  irreg- 
ularly and  disintegrates  them.  Translocation  diastase,  on 
the  contrary,  produces  neither  corrosion  nor  disintegration. 

The  solution  of  starch  takes  place  layer  by  layer;  the 


AMYLASES   OF  DIt-FERENT  SOURCES.  I3£ 

grains  retain  their  original  shape,  but  diminish  gradually  in 
size  as  long  as  they  are  visible. 

This  singular  difference  in  the  mode  of  digestion  appears 
at  first  sight  completely  to  confirm  the  hypothesis  of  the 
existence  of  different  amylases.  But  this  new  argument  ap- 
pears much  less  conclusive  if  we  study  with  more  care  the 
different  modes  of  attack  of  starch  by  diastase.  According 
to  Krabbe,  the  attack  on  the  starch  is  produced  in  a  very  dif- 
ferent manner  in  different  plants.  In  potato-starch  and  in 
that  of  grains  the  digestion  is  accomplished  in  successive 
layers;  the  corrosion  is  centripetal  and  uniform.  In  the 
case  of  leguminous  plants  the  amylase  produces  at  the  sur- 
face of  the  grain  fissures  which  extend  towards  the  center  of 
the  grain  where  they  unite,  forming  a  cavity  which  con- 
stantly increases.  Corrosion  then  takes  place  here  in  two 
directions;  it  is  first  centripetal,  then  it  becomes  centrifugal. 

With  grains,  on  the  contrary,  the  starch  is  unequally  at- 
tacked ;  channels  and  grooves  are  formed  which  extend  to- 
wards the  center. 

These  facts  show  us  that  the  manner  of  digestion  of 
starch  varies  in  different  plants.  And,  in  reality,  the  manner 
of  action  of  amylase  is  very  complex,  even  when  it  is  a  ques- 
tion of  the  digestion  of  grains  of  starch  of  the  same  origin. 
Here  also  is  found  a  very  variable  manner  of  working  and  it 
is  observed  that  the  grains  are  not  all  attacked  in  the  same 
manner.  When  cold  starch  is  treated  with  an  infusion  of 
malt,  digestion  occurs  without  any  regularity.  In  certain 
cells  the  corrosion  is  accompanied  by  clefts  and  holes,  in 
others  the  attack  is  made  in  a  regular  manner.  These  dif- 
ferences evidently  come  from  the  compactness  and  the  non- 
homogeneity  of  the  granules  of  starch.  Moreover,  the  man- 
ner of  digestion  may  also  be  influenced  by  the  reaction  of  the 
medium  as  well  as  by  the  presence  of  foreign  bodies. 

Starch,  difficultly  dissolved  in  the  cold,  easily  digests  in 
slightly  acid  media.  As  the  acid  reaction  favors  but  slightly 
the  saccharification  of  starch,  the  action  observed  on  whole 


136  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

grains  can  only  be  explained  by  the  change  which  the  acid 
produces  in  the  physical  state  of  the  grains ;  it  is  probable 
that  this  acid  reaction  favors  the  contact  of  the  diastase  with 
the  starch. 

The  facts  announced  by  Brown  and  Morris  have  not, 
moreover,  received  sufficient  confirmation. 

It  has  not  yet  been  demonstrated  that  two  diastases  of 
different  origin  always  act  differently  on  raw  starch  and, 
even  if  this  had  been  proved,  one  ought  not  to  conclude  from 
it  that  different  amylases  exist.  The  difference  in  action  of 
various  diastases  might  in  fact  come  from  the  foreign  sub- 
stances which  accompany  them. 

The  artificial  diastase  of  Reichler  is  also  cited  as  consti- 
tuting another  variety. 

This  worker,  on  digesting  gluten  in  a  certain  quantity  of 
slightly  acidified  water,  found  that  the  saccharifying  power 
of  the  liquid  gradually  increased. 

The  enzyme  obtained  in  this  way  shows  all  the  properties 
of  diastases  of  raw  grain,  and  it  is  admitted  that  it  is  formed 
by  the  action  of  the  acid  on  the  gluten. 

According  to  Lintner  the  formation  of  that  enzyme  is  due 
to  a  hypothetical  substance  contained  in  the  gluten,  practi- 
cally a  zymogen,  which  under  the  action  of  acids  is  trans- 
formed into  amylase. 

Malt  amylase  is,  in  fact,  present  in  a  very  small  quantity, 
and  the  increase  of  diastatic  power  results  simply  from  the 
change  in  the  medium  produced  by  the  action  of  the  acid. 

Changes  Produced  in  the  Activity  of  Diastases  at  a 
Temperature  of  700. — By  saccharifying  starch  with  an  in- 
fusion of  malt,  very  different  quantities  of  maltose  are  ob- 
tained according  to  the  temperature  at  which  one  works. 

Analogous  results  are  obtained  by  merely  heating  the  in- 
fusion to  different  temperatures. 

According  to  O'Sullivan,  there  is  for  each  temperature 
a  certain  degree  of  hydration  of  staich  which  is  easily  at- 
tained but  cannot  be  exceeded. 


AMYLASES   OF  DIFFERENT  SOURCES.  137 

The  infusion  heated  to 

K  /would    cause    a    decomposition  \*  maltose  and  >  dextrin. 

DO      >      ,      ,  ,  . .       «  -    I  "  "2  " 

_   o  1  ut  starch  corresponding  to  J 

70    )  ^  &  (1  "     5 

By  keeping  the  diastase  successively  at  temperatures  of 
640,  68°,  and  70°,  a  total  change  is  each  time  produced  in  the 
manner  of  work,  and  it  may  be  concluded  that  either  real 
transformations  of  the  active  substance  have  taken  place,  or 
that  there  is  an  artificial  formation  of  different  types  of  amy- 
lase. 

Our  knowledge  of  the  effect  of  chemical  conditions  upon 
diastases  leads  us  to  quite  another  interpretation. 

The  temperature  has  no  other  effect  than  to  reduce  the 
diastatic  power.  The  nearer  the  temperature  approaches  to 
700,  the  greater  is  this  reduction.  Only,  while  the  diastase 
is  losing  its  real  activity,  there  is  still  an  apparent  activity, 
owing  to  foreign  substances  contained  in  the  infusion,  which 
act  with  a  still  greater  energy  as  the  diastase  becomes 
weaker. 

To  sum  up,  we  have  here  a  phenomenon  which  we  have 
already  observed  concerning  the  diastase  of  raw  grains,  only 
the  action  is  more  complicated  in  the  present  case. 

The  diastase,  maintained  at  a  temperature  of  68°  to  700, 
has  not  the  same  properties  as  the  amylase  of  raw  grains  :  the 
saccharifying  power  has  largely  disappeared  but  the  liquefy- 
ing power  has  not  been  affected. 

From  this  it  results  that  the  heated  infusion,  though  act- 
ing like  the  diastase  of  raw  grains,  differs  from  the  latter  in 
the  ease  with  which  it  liquefies  starch. 

Condition  of  the  Secretion  of  Amylase. — After  having 
studied  the  action  of  physical  and  chemical  agents  on  amy- 
lase, we  will  briefly  discuss  the  method  of  secretion  of  this 
enzyme,  as  well  as  the  conditions  which  favor  its  produc- 
tion. 

In  grain  in  germination,  it  is  the  embryo  alone  which 
plays  an  active  part;  the  role  of  the  endosperm  is  entire! v 
secondary. 


J38  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

The  embryo  of  grains  of  barley,  detached  with  care,  may 
jDe  transformed,  if  it  is  put  in  a  damp  place  and  under  suitable 
conditions  of  temperature,  into  a  little  plant.  The  vegeta- 
tion produced  under  these  conditions  is  very  delicate  and 
short-lived,  but  the  germ  nevertheless  consumes  its  reserve 
food  and  secretes  amylase.  If  the  germ  is  placed  on  its  own 
endosperm  reduced  to  pulp,  the  vegetation  becomes  normal 
and  the  course  of  diastatic  secretion  may  be  followed  by  the 
chemical  transformation  which  is  produced  in  the  amy- 
laceous matter.  By  cultivating  the  germ  in  different  nutri- 
tive media  and  under  different  conditions,  very  interesting 
data  may  be  obtained  upon  the  conditions  which  regulate 
the  secretion  of  diastase. 

Brown  and  Morris,  by  adopting  this  method,  have  made 
some  interesting  discoveries  upon  the  influence  of  different 
carbohydrates  and  of  the  acidity  of  the  medium  upon  the 
production  of  the  diastase. 

By  cultivating  the  same  number  of  embryos,  in  simple 
gelatine  on  the  one  hand,  in  gelatine  with  six  thousandths 
of  a  part  of  formic  acid  added  on  the  other  hand,  they  have 
found  a  noteworthy  difference  in  the  quantities  of  diastase 
secreted. 

Fifty  embryos  cultivated  in  simple  neutral  gelatine  have 
furnished  a  quantity  of  diastase  corresponding  to  o.  118  gr. 
of  oxide  of  copper.  The  diastase  was  found  distributed  in 
the  following  manner:  In  the  germs  0.0708  gr.,  in  the 
gelatine  0.0478  gr.  The  50  germs  cultivated  in  the  acidu- 
lated gelatine  produced  a  quantity  of  diastase  corresponding 
to  0.145  gr.  of  oxide  of  copper.  It  was  distributed  as  fol- 
lows: In  the  germs  0.0904  gr.,  in  the  gelatine  0.0546  gr. 
The  acidity  of  the  medium,  therefore,  clearly  favors  the 
secretion  of  diastase.  By  adding  to  gelatine  different  assim- 
ilable carbohydrates  other  than  starch,  they  have  found  that 
these  substances  act  very  unfavorably  on  the  secretion. 

The  property  of  secreting  diastase  is,  therefore,  not  a 
fundamental  property  of  cells. 


AMYLASES   OF  DIFFERENT  SOURCES.  139 

The  appearance  of  the  diastase  depends  upon  the  method 
of  nutrition,  but  let  us  note,  however,  that  this  appearance 
does  not  always  correspond  to  the  real  needs  of  the  cells, 
and  that  it  must  not  be  considered  as  an  indication  of  in- 
telligence of  the  cells  which,  by  the  aid  of  a  diastatic  secre- 
tion, would  adapt  themselves  to  different  media.  A  barley 
germ  cultivated  in  gelatine  in  which  it  cannot  obtain  nutri- 
tive matters  secretes  the  same  quantity  of  amylase  as  if  it 
were  cultivated  in  starch.  The  secretion  is  always  abundant 
when  the  germ  is  found  in  poor  nutritive  conditions  and  it  is 
checked  as  soon  as  an  assimilable  substance  appears. 

Here,  as  in  the  case  of  sucrase  which  we  studied  above, 
the  secretion  of  diastase  is  a  consequence  of  mal-nutrition, 
and  the  primary  cause  of  all  the  variations  observed  in  the 
secretion  is  nothing  else  than  the  reaction  of  the  medium. 

The  secretion  of  amylase,  as  we  have  just  seen,  is  favored 
by  acidity  of  the  medium.  The  degree  of  acidity  of  the  cel- 
lular substances,  therefore,  influences  considerably  the  inten- 
sity of  the  secretions. 

Starting  with  this  statement,  wre  can  explain  why  secre- 
tion is  favored  where  nutriment  is  lacking.  The  cells,  when 
they  find  non-assimilable  substances  present,  consume  their 
reserves,  and  this  consumption  produces  in  their  interior  a 
change  of  composition  which  favors  osmosis.  The  saline 
substances  of  the  surrounding  medium  then  penetrate  more 
easily  into  the  cells  and,  as  a  result  of  dissociation,  there  is 
produced  an  accumulation  of  acids  which  favor  secretion. 

Analysis  of  Amylase. — The  method  used  to  determine 
the  diastatic  power  of  a  solution  is  based  on  the  following 
observation  of  Kjeldahl : 

As  long  as  the  diastase  is  in  the  presence  of  a  large 
amount  of  non-transformed  starch,  the  quantity  of  malt  pro- 
duced is  proportional  to  the  quantity  of  diastase  contained 
in  the  solution:  in  other  words,  there  is  a  constant  ratio  be- 
tween the  quantities  of  maltose  formed  and  diastase  em- 


J40  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

ployed,  as  long  as  the  latter  acts  in  the  presence  of  a  great 
quantity  of  non-transformed  starch. 

This  observation  has  been  verified  and  confirmed  by  var- 
ious experimenters,  and  it  is  incontestable  that  by  submitting 
various  specimens  of  the  same  starch,  at  the  same  tempera- 
ture, to  the  action  of  increasing  amounts  of  diastase,  quanti- 
ties of  maltose  are  obtained  proportional  to  the  quantities  of 
diastase  used.  The  condition  essential  to  the  success  of  this 
determination  is  that  in  all  the  specimens  a  minimum  amount 
of  diastase  is  used,  an  amount  capable  of  transforming,  at 
the  most,  40  to  50  per  cent  of  the  starch  into  sugar. 

Starting  with  this  principle,  it  is  easy  to  determine  the 
fermenting  power  of  a  liquid.  It  is  sufficient  to  have  a 
standard  diastatic  solution  of  known  value  and  to  make  com- 
parative experiments  with  starch.  A  2  per  cent  solution  of 
soluble  starch  is  generally  used. 

To  100  cubic  centimetres  of  solution  containing  2  grams 
of  starch  add  2  cubic  centimetres  of  a  solution  of  standard 
amylase.  In  another  dish  also  containing  100  cubic  cen- 
timetres of  a  solution  of  soluble  starch  add  2  cubic  centime- 
tres of  the  solution  to  be  tested.  Place  the  two  specimens, 
in  a  water-bath  at  500,  and,  after  an  hour  of  saccharification, 
measure  the  maltose  in  the  two  solutions. 

The  diastatic  power  of  the  solution  is  expressed  by  the 
ratio  between  the  quantities  of  sugar  formed  with  equal 
quantities  of  the  experimental  diastatic  solution  and  the 
standard  solution. 

If  there  is  found,  for  example,  0.4  of  maltose  in  the  prod- 
uct saccharified  with  standard  amylase  and  0.2  of  maltose  in 
the  second  specimen,  we  should  say  that  the  activity  of  the 
solution  is  50  per  cent,  meaning  that  the  solution  examined 
is  half  as  active  as  the  standard  liquid. 

This  method  of  analysis  permits  a  comparison  of  the 
value  of  two  products,  but  it  does  not  permit  the  absolute  ex- 
pression of  the  fermenting  power  of  a  diastase,  because  it  is 
very  difficult  to  maintain  in  a  solution  of  amylase  a  constant 


AMYLASES   OF  DIFFERENT  SOURCES.  14* 

diastatic  energy.  The  results  are,  therefore,  often  uncer- 
tain. 

To  determine  absolute  diastatic  values,  we  use  a  method 
in  which  we  take  for  unity  the  quantity  of  diastase  which, 
acting  for  an  hour  at  6o°  on  i  gram  of  soluble  starch,  gives 
50  centigrams  of  maltose. 

The  following  is  the  regular  procedure: 

Ten  grams  of  anhydrous,  neutral  starch  are  dissolved  in 
700  cubic  centimetres  of  boiling  water.  It  is  cooled  and  the 
volume  of  the  solution  brought  up  to  750  c.c.  From  this 
solution  are  taken  a  series  of  specimens  of  75  cubic  centime- 
tres each.  To  these  specimens  are  added  different  quanti- 
ties of  the  active  liquid  to  be  examined  and  they  are  left  for 
an  hour  in  a  water-bath  at  6o°.  The  saccharification  finished, 
all  the  specimens  are  rapidly  brought  up  to  the  boiling  point, 
cooled,  brought  up  to  100  cubic  centimetres  and  in  each  of 
them  the  quantity  of  sugar  produced  is  determined.  The 
specimen  in  which  50  centigrams  of  maltose  is  formed  is  re- 
garded as  the  standard  unit.  If  these  50  centigrams  are 
formed  in  the  specimen  to  which  was  added  1  cubic  centime- 
tre of  the  solution  experimented  with,  we  say  that  the  dia- 
static power  of  that  solution  is  100.  If  these  50  centigrams 
are  found  in  the  tube  to  which  2  cubic  centimetres  of  the 
solution  were  added,  we  say  that  the  diastatic  power  is  50, 
and  so  on. 

It  is  often  difficult  with  a  single  series  of  experiments  to 
succeed  in  producing  exactly  %  gram  of  maltose.  So  it  is  of 
advantage  to  make  first  an  approximate  experiment  with  1, 
2,  4,  6,  8,  10  cubic  centimetres  of  active  substance.  If  the 
unit  of  diastatic  activity  is  approximated,  for  example,  in  the 
experiment  made  with  4  cubic  centimetres  of  infusion,  we 
repeat  the  experiments  with  2.5,  2.75,  3,  3.25,  3.50,  3.75  c.c. 
of  liquid. 

One  must  also  take  into  account  in  these  experiments 
the  quantity  of  reducing  substances  which  may  be  found  in 
the  active  solution.     One  must  of  course  subtract  from  the 


142  THE  ENZYMES  AND    THEIR   APPLICATIONS. 

total  quantity  'of  maltose  found  after  saccharification  the 
quantity  of  sugar  which  was  introduced  with  the  infusion. 

This  method  may  also  be  applied  to  an  analysis  of  malt. 

To  estimate. the  diastatic  power  of  malt  we  must  first  ex- 
tract the  active  substances.  For  that  we  reduce  the  malt  to 
fine  powder,  add  20  parts  of  water  and  leave  it  for  6  hours  at 
a  temperature  of  300,  shaking  the  solution  every  quarter  of 
an  hour.  With  the  filtered  infusion,  saccharification  is  ac- 
complished as  has  just  been  indicated.  A  malt  of  excellent 
quality  affords  under  these  conditions  an  infusion  producing 
50  centigrams  of  maltose  per  cubic  centimetre  of  infusion. 
Still,  this  method  does  not  furnish  precise  data  on  the  value 
of  a  malt  from  a  practical  point  of  view.  In  the  chapter 
treating  of  the  industrial  applications  of  amylase,  we  shall 
treat  particularly  of  such  analyses. 

The  determination  of  the  saccharifying  power  of  liquids 
containing  slight  amounts  of  amylase  often  presents  great 
difficulties.  To  obtain  an  appreciable  quantity  of  maltose,  it 
is  necessary  to  use  a  great  quantity  of  solution  which  often 
contains  reducing  materials. 

In  such  cases  it  is  better  first  to  precipitate  the  diastase 
by  alcohol,  but  this  method  is  applicable  only  when  one  has 
a  quite  considerable  volume  of  solution  at  his  disposal,  for 
when  this  precipitation  is  practiced  on  a  small  quantity  of  in- 
fusion, a  very  fine  precipitate  is  obtained  which  passes 
thiough  the  filter  and  consequently  gives  rise  to  perceptible 
losses.  To  remedy  this  difficulty,  we  have  sought  to  pro- 
duce  in  the  active  solution  precipitates  which  are  more 
voluminous  and  more  easily  separated.  We  have  found  that 
tannin  leads  to  this  result.  In  fact  our  experiments  have 
shown  us  that  this  substance  completely  precipitates  the  dia- 
stase and  that  the  inactive  precipitate  becomes  active  again 
when  it  is  carefully  treated  with  a  dilute  solution  of  sodium 
•carbonate. 

The  method  of  procedure  is  here  given : 

To  10  cubic  centimetres  of  active  liquid  add  4  centigrams 


AMYLASES   OF  DIFFERENT  SOURCES.  143 

of  tannin  dissolved  in  a  few  cubic  centimetres  of  water;  stir 
it  and  leave  it  for  a  half-hour.  The  solution  is  then  filtered 
and  the  precipitate,  well  washed  with  water  and  alcohol,  is 
placed,  without  being  separated  from  the  filter,  in  a  glass 
capsule  containing  5  cubic  centimetres  of  sodium  carbonate 
(1  :  10,000).  The  filter  is  washed  in  the  solution  for  one  or 
two  minutes;  as  soon  as  the  precipitate  is  redissolved  add  a 
few  drops  of  a  solution  of  lactic  acid  (1 :  1000)  to  neutralize  it, 
and  filter. 

All  these  manipulations  must  be  made  as  rapidly  as  pos- 
sible because  the  tannin  precipitate  changes  by  a  prolonged 
exposure  to  the  air  and  becomes  insoluble  in  the  alkaline 
-solution.  The  contact  of  the  precipitate  with  the  carbonate 
of  sodium  must  also  be  of  very  short  duration.  When  the 
precipitate  does  not  redissolve  except  after  4  or  5  minutes  of 
contact,  the  experiment  must  be  repeated  because  the  dia- 
stase is  already  changed.  The  solution  can  be  greatly  facili- 
tated by  triturating  the  filter  in  a  mortar  with  the  alkaline 
solution.  By  working  rapidly  it  is  possible  to  redissolve  all 
the  active  substances  precipitated  and  avoid  all  loss.  The 
precipitate  obtained  by  the  tannin,  washed  in  water,  alcohol, 
and  ether,  and  then  dried,  gave  on  analysis  the  following 
figures,  a  deduction  being  made  of  2,2.2%  of  tannin  : 

Water 5.53% 

Nitrogen 8.83 

Ash 1.32 

This  method  is  of  special  use  when  one  wishes  to  deter- 
mine amylase  in  vegetable  cells. 

In  analyses  of  this  kind  the  substances  are  reduced  to 
powder.  They  are  left  to  soak  in  1  to  2  parts  of  water  for 
6  hours;  the  liquid  is  expressed  from  the  substances  not  dis- 
solved. The  residue  is  again  soaked  with  a  volume  or  two 
of  water  and  expressed  a  second  time.  The  combined 
liquids  are  filtered  and  the  diastase  is  precipitated  from  the 
mixture  by  tannin,  in  the  same  way  as  with  the  malt  infusion. 


144  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

The  activity  of  the  precipitate  dissolved  in  the  water 
gives  an  idea  of  the  diastatic  value  of  the  substances  sub- 
mitted to  examination. 

Here,  for 'example,  is  an  analysis  of  bean-leaves: 
Ten  grams  of  ■  bean-leaves  are  reduced  to  a  paste  in  a 
mortar.  Ten  cubic  centimetres  of  water  is  added  to  the  mass 
and  a  few  drops  of  chloroform,  then  it  is  allowed  to  stand  for 
6  hours.  The  leaves  are  then  pressed  and  filtered  in  a  cloth. 
To  the  residue  is  then  added  10  cubic  centimetres  of  water 
and  a  small  drop  of  chloroform,  after  which  the  whole  is  left 
quiet  for  three  hours.  Then  the  liquid  is  separated  and  the 
residue  washed  again  with  water;  the  liquids  of  the  two 
macerations  and  of  the  washing  are  combined,  and  the  total 
volume  made  up  to  50  cubic  centimetres.  It  is  again  filtered 
and  precipitated  with  16  cubic  centimetres  of  tannin.  The 
precipitate  is  redissolved  in  alkaline  water  and  the  solution 
is  made  up  to  10  cubic  centimetres.  It  is  found  necessary  to 
use  2  cubic  centimetres  of  this  solution  to  produce  50  milli- 
grams of  maltose.  Therefore,  the  solution  has  a  diastatic 
power  of  50.  If  we  compare  the  diastatic  power  of  bean- 
leaves  with  that  of  a  good  quality  of  malt  we  shall  obtain  the 
following-  results :  Ten  grams  of  malt  furnish  200  cubic  cen- 
timetres of  infusion,  one  centimetre  of  which  produces  50 
milligrams  of  maltose.  Ten  grams  of  bean-leaves  furnish  10 
cubic  centimetres  of  liquid  of  which  2  cubic  centimetres  are 
needed  to  furnish  50  milligrams  of  maltose.  Since  the  malt 
has  a  diastatic  power  of  100,  the  bean-leaves  have  one  of  2.5. 
The  malt  contains  consequently  40  times  as  much  active  sub- 
stance as  the  bean-leaves. 

BIBLIOGRAPHY. 

Sig.    Kirchoff. — Ueber   die    Zuckerbildung   beim    Malzen    des    Getreides. 

Schweiggers  Journal,  1815,  p.  389. 
Dubrunfaut. — Memoire  sur  la  sacchariflcation  des  fecules,  2e  edition,  Gau- 

thier-Villars,  Paris,  1882. 
Guerin  Varry. — Memoire  concernant  Taction  de  la  diastase  sur  l'amidon 

de  pomme  de  terre.     Ann.  de  chimie  et  de  phys.,  1835,  p.  32. 


AMYLASES   OF  DIFFERENT  SOURCES.  M5 

Leuchs. — Ueber    die    Verzuckerung    des    Starkemehles    durch    Speichel. 

Kastners  Archiv.  fur  die  Ges.  Naturlehre,  1831. 
Biot. — Memoire  sur  l'amidon.     Ann.  des  Sciences  nat.,  1838,  p.  5. 
Clement  Desormes. — Ann.  de  chimie  et  phys.,  IV,  p.  473. 
Payen  et  Persoz. — Memoire  sur  la  diastase  et  les  principaux  produits  de 

sa  reaction.     Ann.  de  chimie  et  phys.,  1833. 
Miahle. — De  Taction  de  la  salive  sur  l'amidon.     Comptes  Rendus,  XX, 

1845,  p.   1485. 
De  la  digestion  et  de  Tassimilation  des  matieres  sucrees.     Comptes 

Rendus,  1845,  p.  954,  t.  XX. 
Musculus. — Sur  la  transformation  de  la  matiere  amylacee  en  glucose  et  en 

dextrine.     Ann.  de  chimie  et  de  phys.,  i860,  LX,  p.  203. 
Bouchardat  and  Sandras. — Des  fonctions  du  pancreas  et  de  son  influence 

sur  la  digestion  des  fecules.     Comptes  Rendus,  1845,  XX,  p.  1085. 
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Chemical  Society,  1872-1874. 
Kossmann. — Recherches   chimiques   sur  les  ferments  contenus  dans   les 

vegetaux.     Bull,  de  la  Soc.  chim.  de  Paris,  1877. 
Baranetsky.— Die  Starke  umwandelnder  Fermente,   1878. 
Ch.   Richet. — Du   sue  gastrique  chez  l'homme  et  les  animaux.     These, 

Paris,  1878. 
Kjeldahl. — Recherches    sur   le    ferment    producteur    du    sucre.     Comptes 

Rendus  des  trav.  du  laboratoire  de  Carlsberg,  1879. 
Brown  and  Heron. — Beitrage  zur  Geschichte  der  Starke  und  der  Ver- 

wendung  derselben.     Liebig's  Annalen,   1879. 
Brown  and  Morris. — Journal  of  the  Chem.  Soc,  1890. 
J.  Lintner. — Studien  iiber  Diastase.     Journ.  f.  prakt.  Chemie,  1886,  p.  378. 
Ueber  das  diastatische  Ferment  des  ungekeimten  Weizen.     Zeit.  fur 

das  ges.  Brauwesen,  1888. 
Em.  Bourquelot. — Sur  la  separation  et  le  dosage  du  glycogene  dans  les 

tissus.     Journal  des  connaissances  med.,  1884. 
Chittenden   and    Smith. — The   diastatic    action    of   saliva   as    modified   by 

various  conditions,   studied  quantitatively.     Chemical    News,    1886. 
Brown  and   Morris. — Untersuchung  iiber  die    Keimung  einiger   Graser. 

Zeit.  fur  das  gesammte  Brauwesen,  1890. 
Moritz   and    Glandening. — Sur    Taction    de    la    diastase.      The    Chemical 

Society,  1892. 
Lintner  and  Dull. — Ueber  den  Abbau  der  Starke  unter  dem  Einfluss  der 

Diastasewirkung.   Berichte  der  deutschen  chem.  Gesellsch.,   1893,  p. 

2533- 

Sohiffer. — Sur  les  produits  incristallisables  de  Taction  de  la  diastase  sur 
l'amidon.     Moniteur  scientifique,   1893,  p.  712. 

J.  V.  Egoroff. — Sur  la  diastase  des  grains  crus.  Journal  de  la  Soc.  de 
chimie  et  phys.  Saint-Petersbourg,  1893,  t.  XXV. 

Effront. — Contribution  a  Tetude  de  la  saccharification.  Moniteur  scien- 
tifique. 


146  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

Effront. — Actions    des   acides    mineraux   dans    la    saccharification    par   le 

malt.     Moniteur  scientifique,   1890. 
Sur  les  conditions  chimiques  de  Taction  des  diastases.     Comptes  Ren- 

dus,  1892,  p.  1324. 

■ Sur  l'amylase.     Comptes  Rendus,  1895. 

L'Influence    des    antiseptiques    sur    les    ferments.     Moniteur    scien- 
tifique,  1894. 
Contribution    a    l'etude    de    l'amylase.     Moniteur    scientifique,    1895, 

VIII,  p.  54i;  X,  711. 
Henri   Pottevin. — Sur  la  saccharification   de  l'amidon   par  l'amylase  du 

malt.     Comptes  Rendus,  1898,  p.  17. 
Duclaux. — Sur  la  saccharification.     Ann.  de  l'lnst..  Pasteur,  1895,  56. 

• Les  theories  de  la  saccharification.     Ann.  de  l'lnst.  Pasteur,  1895,  170.' 

■  Amidon,  dextrine  et  maltose.     Ann.  de  l'lnst.  Pasteur,   1895,  p.  215. 

Brown  and  Morris. — Einwirkung  der  diastase  auf  Starke.     Berichte  der 

deutschen  chemischen  Gesellschaft,  1895,  p.  642. 
H.    Seyffert. — Untersuchungen    fiber    Gerste    und    Malz    Diastase.     Zeit- 

schrift  fur  das  gesammte  Brauwesen,  1898. 
A.  Wroblewsky. — Ueber  die  chemischen  Eigenschaften  der  Diastase  und 

fiber   das   Vorkommen    eines   Arabans   in    der   Diastase    Preparaten. 

Berichte   der  deutschen  chem.   Gesellsch.,   1897,  2,   p.  2289;   1897,  3, 

p.  3048. 
Osborne    and    Campbell. — Wirkung    der     Diastase    bei    fortschreitender 

Keimung.     Berichte,  1896,  p.  1159,  Journal  Amer.  Chem.  Soc,  18,  p. 

536-542. 
O.  Nass  and  Framm. — Bemerkungen  zur  Glycolyse.     Pflug.  Archiv,  63,  p. 

203-208. 
A.  Sing  and  Baker. — Journal   Chem.  Soc,  67,  p.  702-708. 


CHAPTER  XI. 

INDUSTRIAL  APPLICATIONS   OF   AMYLASE. 

Malting. — Chemical    transformations    which    accompany    germination. — ^ 
Methods  of  malting,  sorting,  steeping,  germination,  brewing. 

Amylase  is  formed  in  considerable  quantities  in  the  grains 
of  cereals  during  germination.  It  is  for  this  reason  that  the 
industries  which  utilize  diastase  as  an  agent  of  hydration 
make  use  of  sprouted  grain'  called  malt,  a  product  which  at 
the  present  time  is  the  "only  agent  of  that  kind  capable  of 
being  manufactured  economically.  All  cereals  produce  amy- 
lase during  germination,  but  barley  furnishes  the  greatest 
yields  in  active  substance.  When  one  heaps  up  barley  pre- 
viously soaked,  one  observes  a  series  of  phenomena  which, 
all  together,  characterize  germination.  First  is  found  an  in- 
crease in  temperature,  an  absorption  of  oxygen,  and  a  libera- 
tion of  carbonic  anhydride,  which  is  augmented  as  the  tem- 
perature of  the  mass  increases.  Along  with  this  phenom- 
enon of  respiration  are  observed  considerable  changes  in 
the  various  constituents  of  the  grain.  The  reserve  materials, 
cellulose,  starch,  protein  matters  and  fatty  substances  as  well 
as  sugars  are  partially  transformed  by  hydration. 

These  transformations  are  due  to  a  secretion  of  enzymes 
acting  on  the  albumen  and  transforming  it  into  assimilable 
substances,  which  are  in  part  absorbed  by  the  embryos  in  the 
course  of  their  development. 

After  24  or  48  hours  of  germination,  there  are  seen  to 
appear,  on  the  outside  of  the  grains,  little  roots  which  then 
grow  quite  rapidly.  The  development  of  the  plumule  is  much 

147 


14§  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

slower.  After  8  or  10  days  of  germination,  the  length  of  the 
plumule  reaches  half  or  three  quarters  that  of  the  grain.  It 
is  at  this  phase  of  development  that  germination  is  generally 
considered  as  ended. 

The  development  of  the  germ  is  largely  at  the  expense 
of  the  starch.  Under  normal  conditions  the  expenditure  in 
amylaceous  materials  is  8  to  10  per  cent  of  the  starch  con- 
tained in  the  grains,  but  this  proportion  is  considerably  ex- 
ceeded when  germination  is  accomplished  at  a  temperature 
higher  than  200. 

During  germination  the  grain  secretes,  besides  amylase, 
other  active  substances,  among  them  peptase,  which  trans- 
forms albuminoid  substances  into  amides,  and  cytase  which 
acts  on  certain  kinds  of  cellulose. 

The  role  of  cytase  is  very  important  from  the  point  of 
view  of  malting. 

The  starch  in  the  grains  is  in  the  form  of  granules 
enclosed  within  resistant  cell-walls.  These  cell-walls  pro- 
tect the  starch  against  the  action  of  the  amylase,  and  the  at- 
tack on  the  carbohydrate  would  not  be  very  strong  without 
the  intervention  of  the  cytase  which  disintegrates  the 
envelope  of  the  granules. 

The  action  on  the  starch,  during  germination,  is  accom- 
plished in  two  successive  phases.  In  the  first  phase  the  cel- 
lulose envelopes  of  the  starch  cells  are  liquefied  by  cytase, 
and  it  is  then  only  that  the  amylase  begins  to  act  on  the 
starch.  To  the  action  of  cytase  must  also  be  attributed  the 
differences  found  between  the  starch  of  raw  grains  and  the 
starch  of  malt. 

On  account  of  the  destruction  of  the  membrane  of  the 
cell  the  malt-starch  is  liquefied  at  a  lower  temperature  than 
the  starch  of  raw  grains. 

When  germination  is  produced  at  a  temperature  of 
15-170,  the  secretion  of  amylase  commences  after  35  or  40 
hours  and  the  diastatic  power  then  gradually  increases  for  8 
or  10  days. 


INDUSTRIAL   APPLICATIONS   OF  AMYLASE.  149 

In  the  practice  of  malting  the  grains  are  submitted  to  a 
succession  of  operations. 

The  first  part  of  the  work  consists  in  sorting  and  cleans- 
ing the  grains.  Then  they  are  soaked,  then  allowed  to  ger- 
minate. The  germinated  grains  are  utilized  in  a  fresh  state 
in  distilleries  as  well  as  in  the  manufacture  of  maltose;  for 
the  purposes  of  the  brewery  the  sprouted  grains  are  malted. 

Without  entering  into  all  the  details  of  these  different 
manipulations,  let  us  consider  the  principal  points. 

The  sorting  of  the  grains  is  done  in  special  apparatus 
which  eliminates  foreign  substances  as  well  as  broken  grains. 
Moreover,  this  apparatus  separates  the  grains  according  to 
their  dimensions. 

The  grain  intended  for  germination  must  not  be  too 
fresh.  Grain  taken  immediately  after  the  harvest  has  a  small 
germinating  power.  It  is  only  after  some  time  that  it  be- 
comes good  for  germination.  Grain  coming  from  different 
harvests  must  not  be  mixed,  nor  grain  having  different  den- 
sities. To  obtain  a  good  germination  it  is  really  indispen- 
sable that  the  grains  should  be  as  far  as  possible  of  a  uniform 
weight. 

In  sorting,  the  grains  are  separated  according  to  their 
sizes.  Grains  of  different  sizes  could  not  be  put  together  in 
germination  because  they  would  soak  unequally. 

Grains  of  a  different  weight  are  not  suitable  for  the  same 
use.  Heavy  grains  are  preferable  for  use  in  the  brewery, 
while  light  grains,  containing  less  starch  and  furnishing  a 
much  greater  yield  in  diastase,  are  suitable  rather  for  the 
work  of  the  distillery. 

The  sorted  barley  is  then  steeped.  This  operation  is 
generally  carried  on  in  special  vats  which  easily  permit  of 
changing  the  water.  The  aim  of  the  soaking  is  to  make  the 
grains  absorb  the  quantity  of  water  necessary  for  a  good  ger- 
mination. The  grains,  in  contact  with  water,  swell,  absorb 
a  certain  amount  of  oxygen,  and  undergo  different  modifica- 
tions. 


150  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

They  also  lose  a  part  of  their  soluble  substances,  espe- 
cially salts  and  carbohydrates  other  than  starch.  The  loss  in 
extractive  substances  varies  from  0.8  to  1  per  cent. 

The  elimination  of  sugar  by  the  soaking  of  the  grains  is 
very  favorable  to  the  secretion  of  the  enzymes  during  ger- 
mination. 

In  ordinary  water,  there  is  not  sufficient  oxygen  for 
normal  germination,  so  it  is  advisable  to  pass  a  current  of  air 
into  the  mass  during  the  soaking. 

The  soaking  water  must  be  frequently  renewed  so  that" 
the  dissolved  substances  shall  not  enter  into  fermentation. 

Generally,  the  grains  are  washed  in  water  before  soaking 
them  to  free  them  from  germs  and  ferments  which  may 
adhere  to  their  surface. 

The  grains  are  left  in  water  for  3  to  5  days,  and  the  water 
is  carefully  renewed  every  12  or  24  hours.  The  duration  of 
the  soaking'  depends  upon  many  factors ;  it  depends  on  the 
temperature  as  well  as  the  quality  of  the  water,  but  it  de- 
pends especially  on  the  quality  of  the  grains.  Thick-glumed 
barley  absorbs  water  more  slowly  than  thin-glumed  grain. 

The  operation  of  soaking  the  grains  may  be  considered 
as  ended  when  they  have  absorbed  nearly  50  per  cent  of 
water.  By  prolonging  the  soaking  the  grains  would  absorb 
a  still  greater  quantity  of  water,  but  in  that  case  germination 
would  be  less  regular  and  there  would  be  danger  of  getting 
mouldy  malts.  It  is  very  difficult  to  stop  the  soaking  at 
exactly  the  right  point.  This  difficulty  comes  from  the  dif- 
ferences in  the  grains  employed.  It  is,  therefore,  ad- 
visable to  stop  the  process  before  the  grains  are  sufficiently 
soaked.  The  danger  of  soaking  too  long  is  especially  great 
when  rye  is  used.  The  grains  having  had  too  long  a  soak- 
ing become  sticky,  acquire  a  pasty  aspect,  and  the  malt  they 
furnish  is  of  a  doubtful  quality. 

The  grains,  after  having  been  soaked,  are  carried  to  the 
malt-house,  where  they  are  spread  in  layers  of  30  to  80  cen- 
timetres in  depth,  according  to  the  kind  of  malt-house  and 


INDUSTRIAL   APPLICATIONS   OF  AMYLASE.  I51 

the  manner  of  aeration.  Malt-houses  must  conform  to  the 
two  following  conditions : 

They  must  be  (1)  well  ventilated,  and  (2)  capable  of  being 
kept  at  a  constant  temperature. 

The  heaped-up  grains  heat  quite  rapidly.  The  oxidation 
of  starch  and  fatty  materials  frees  a  quantity  of  heat  sufficient 
to  bring  the  entire  mass  to  a  temperature  of  ioo°.  It  is  then 
necessary  to  avoid  raising  the  temperature.  This  is  accom- 
plished, either  by  frequently  changing  the  position  of  the 
grains,  or  by  spreading  them  in  thinner  and  thinner  layers  as 
the  action  becomes  more  energetic.  In  the  system  called 
"  pneumatic  "  the  layers  are  cooled  by  a  current  of  moist  air. 

Germination  lasts  from  8  to  10  days.  It  is  desirable  to 
work  always  at  the  lowest  possible  temperature.  Generally 
the  germination  is  begun  at  a  temperature  of  10-110  and  is 
continued  up  to  17-180,  which  limit  is  not  passed. 

When  the  malt  is  spread  on  a  cement  floor,  the  layers  are 
made  at  the  beginning  of  40  to  50  centimetres  in  thickness, 
and  then  are  progressively  made  thinner.  The  fourth  day  a 
thickness  of  10  to  12  centimetres  is  reached.  In  the  pneu- 
matic system  the  depth  of  the  layers  remains  constant,  but 
the  grain  is  often  turned  over  to  prevent  the  little  roots  from 
tangling. 

During  germination  the  moisture  of  the  grains  constantly 
diminishes  and  at  the  end  of  the  operation  they  have  lost 
from  50  to  60  per  cent  of  the  water  which  they  have  ab- 
sorbed during  the  soaking.  It  often  happens  that  the  water 
absorbed  during  soaking  is  insufficient  to  ensure  germina- 
tion. In  this  case  the  layers  must  be  sprinkled  from  the 
third  or  fourth  day.  The  sprinkling  is  done  systematically 
in  small  quantities  and  at  frequent  intervals.  Generally  the 
germination  is  checked  when  the  length  of  the  plumules 
reaches  half  or  three  quarters  that  of  the  grains. 

It  is  generally  assumed  that  at  this  time  the  grains  con- 
tain the  greatest  quantity  of  active  substances.  In  reality  it 
is  not  so.     The  researches  which  we  have  made  in  this  sub- 


J52 


THE  ENZYMES  AND    THEIR  APPLICATIONS. 


ject  show  that  one  cannot  trust  to  the  length  of  the  plumules 
to  determine  the  time  when  the  quantity  of  diastase  con- 
tained in  the  grains  reaches  its  maximum,  and  that  it  is  only 
analysis  which  can  show  when  germination  should  be 
checked. 

The  following  table  traces  the  course  of  germination,  at 
i2°-iy°,  of  four  different  malts  conducted  under  the  same 
conditions. 


At  the  beginnin 

1  day 

2  days 

3  "    

4  "    

5  "    

6  "    

7  "     

8  "    

io     "    


Diastatic  Power. 


41 
50 
60 
60 
70 
81 
85 
95 
100 
96 


60 

70 

95 

95 

97 

95 

98 

100 

100 

100 


52 
70 
80 
81 
85 
87 
88 
86 
89 
85 


35 
40 

57 
62 
80 
85 
97 
100 

94 


These  experiments,  as  well  as  a  very  great  number  of  ob- 
servations made  in  different  manufactories,  have  led  us  to 
the  following  conclusion :  It  is  when  the  malt  possesses 
plumules  twice  as  long  as  the  grains  that  the  diastatic  power 
reaches  its  maximum;  however,  in  some  cases  the  maximum 
is  not  reached  at  this  time. 

The  quantity  of  diastase  contained  in  the  grain  increases 
gradually  in  the  course  of  germination ;  but  often  reaches  its 
maximum  before  the  plumules  have  reached  the  length  in- 
dicated above. 

The  quantity  of  diastase  developed  in  the  malt  often  re- 
mains stationary  for  a  certain  time. 

In  other  cases,  on  the  contrary,  a  very  rapid  diminution 
of  the  quantity  of  diastase  is  observed.  This  diminution 
may,  in  fact,  be  observed  in  the  table  reproduced  above.  We 
have  sought  the  cause  of  this  decrease  and  have  found  that  it 


INDUSTRIAL   APPLICATIONS   OF  AMYLASE.  153 

comes  from  the  energetic  oxidation  which  occurs  when  ger- 
mination is  very  far  advanced.  It  is  really  in  the  pneumatic 
malt-houses  that  diminution  of  diastase  is  most  frequently 
found,  while  in  common  malt-houses  the  alteration  of  the 
diastase  is  much  rarer.  It  may  be  that  in  addition  to  the 
oxygen  of  the  air  other  factors  also  come  into  play  to 
produce  the  diminution  of  the  diastatic  power  of  the  malt. 

When  a  very  active  malt  is  desired,  it  is  indispensable  that 
it  shall  be  analyzed  after  the  eighth  or  ninth  day,  and  the 
variations  of  its  diastatic  power  observed  twice  a  day.  It  is 
only  in  this  way  that  losses  of  diastase  can  be  avoided. 

In  the  brewery,  fresh  malt  cannot  be  employed.  To 
make  it  suitable  for  the  manufacture  of  beer  it  must  pass 
through  the  malt-kiln  where,  under  the  influence  of  a  high 
temperature,  certain  principles  contained  in  the  grains  un- 
dergo transformations  which  give  to  the  malt  a  characteris- 
tic flavor,  as  well  as  a  more  or  less  dark  color. 

The  drying  is  done  by  the  aid  of  hot  air  and,  according  to 
the  kind  of  malt  which  it  is  proposed  to  make,  the  drying  is 
accomplished  at  higher  or  lower  temperatures. 

The  fundamental  principle  of  malting  consists  in  raising" 
the  temperature  gradually,  especially  at  the  beginning  of 
drying. 

Although  the  grain  contains  from  10  to  12  per  cent  of 
water,  it  is  extremely  dangerous  to  go  above  a  temperature 
of  500.  In  fact  the  malt  diastase  changes  under  the  action 
of  heat  and  this  change  is  the  more  rapid  as  the  grain  con- 
tains a  greater  quantity  of  water. 

The  grains  dehydrated  below  500  may  then  be  brought 
up  to  a  temperature  of  ioo°  without  completely  destroying; 
the  diastase. 

The  highest  temperature  reached  during  the  malting  is 
from  1030  to  1040  for  malt  of  the  Munich  type  and  only  from 
62  °  to  63°  for  malt  of  the  Pilsen  type. 

Drying  always  destroys  a  part  of  the  diastase,  even  when 
all  possible  precautions  are  taken.     In  drying  the  malt  at  the 


154  THE  ENZYMES  AND   THEIR  APPLICATIONS. 

maximum  temperature  of  500  and  avoiding  raising  the  tem- 
perature at  the  beginning,  we  have  found  that  nearly  20  per 
cent  of  active  substances  are  destroyed  during  the  process. 
The  loss  is  hence  seen  to  be  considerable. 

Finally,  there  exists  a  great  difference  between  the  dis- 
tillery malt  and  brewery  malt. 

As  we  have  said  above,  it  is  well  to  choose  for  brewing- 
malt,  grain  which  is  very  heavy  and  very  rich  in  starch.  For 
the  distillery,  on  the  other  hand,  light  grain  which  furnishes 
more  diastase  is  to  be  preferred.  The  germination  of  brew- 
ing-malt should  be  arrested  when  the  plumules  have  acquired 
half  or  three  quarters  the  length  of  the  grains.  When,  how- 
ever, it  is  a  question  of  distillery  malt  one  should  allow  the 
plumule  to  grow  as  long  as  possible. 

Brewery  malt  may  be  aerated  up  to  the  last  moment, 
while  for  distillery  malt,  aeration  should  cease  during  the  last 
two  or  three  days. 

Finally,  there  is  a  great  difference  in  the  drying  of  malt 
according  as  it  is  destined  for  the  brewery  or  the  distillery. 
For  the  distillery  the  temperature  must  be  the  lowest  pos- 
sible, while  for  the  brewery  it  must  be  quite  high. 

BIBLIOGRAPHY. 

Moritz  and   Morris. — Handb.   d.    Brauwissenschaft. 

Prior. — Chemie  und  Physiologie  des  Maizes  und  des  Bieres. 


•  CHAPTER  XII. 

ROLE  OF  AMYLASE  IN  THE  BREWERY. 

The  brewing  industry  was  at  first  carried  on  by  following 
empirical  methods,  and  it  is  only  within  thirty  years  that  the 
manufacture  of  beer  has  drawn  the  attention  of  investigators. 
The  works  of  Pasteur,  Dubrunfaut,  and  Hansen  have 
brought  to  this  domain  valuable  data  which  form  at  the 
present  time  the  scientific  basis  of  this  industry.  The  re- 
searches of  these  workers  have  brought  about  noticeable  im- 
provements in  the  methods  of  manufacturing  beer. 

It  must  be  recognized  that  at  the  present  time  empirical 
methods  have  not  entirely  disappeared  from  the  business  of 
the  brewery,  and  that  science  cannot  yet  explain  all  the 
phenomena  observed  in  the  manufacture  of  beer.  To  carry 
out  this  manufacture  successfully,  it  still  requires  more  prac- 
tice than  science. 

The  brewery  uses  as  raw  materials  malt,  hops,  water,  and 
yeast.  With  these  simple  materials  an  almost  infinite  variety 
of  fermented  beverages  is  made.  The  variations  in  beers 
come,  in  the  first  place,  from  differences  in  the  quality  of  the 
raw  materials.  Brewing-malt  is  far  from  being  a  substance 
of  constant  composition.  It  varies  according  to  the  origin 
and  quality  of  the  barley,  and  also  according  to  the  method 
of  malting  employed.  The  same  is  true  of  the  other  factors 
which  enter  into  the  manufacture  of  beer.  In  fact,  different 
yeasts  act  very  differently  in  the  same  mash  and  give  very 
different  results. 

155 


I56  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

The  difference  in  the  character  of  the  beers  may  also  be 
influenced  by  the  quality  of  the  water  or  that  of  the 
hops. 

The  taste  and  the  appearance  of  the  fermented  product 
may  also  change  on  account  of  the  intervention  of  bacteria 
and  of  foreign  yeasts.  All  these  causes  undoubtedly  influ- 
ence the  manufacture,  but  variation  in  raw  materials  does 
not  explain  all  the  differences  observed  among  fermented 
beverages. 

The  character  of  a  beer  depends  in  reality  on  a  number  of 
factors :  on  the  method  of  work,  the  manner  in  which  the 
malting  and  brewing  are  conducted,  the  methods  of  extrac- 
tion and  saccharification,  as  well  as  the  mode  of  fermenta- 
tion. 

As  may  be  seen,  brewing  is  an  exceedingly  complicated 
industry.  To  understand  the  process,  a  very  complete  scien- 
tific knowledge  is  necessary,  and  even  then  one  often  finds 
problems  which  have  not  been  scientifically  solved.  Fortu- 
nately, the  brewer  solves  the  difficulty  by  observation  as  well 
as  by  the  routine  he  has  acquired.  Malt  is  generally  very  ricfL 
in  enzymes  and  the  amylase  it  contains  can  hydrate  10  to  20 
times  as  much  amylaceous  matter  as  the  malt  contains. 
Liquefaction  and  saccharification  take  place  without s  diffi- 
culty when  there  is  a  great  deal  of  diastase.  If  complete  sac- 
charification were  the  sole  object  sought,  the  problem  would 
be  easily  solved.  But,  in  reality,  the  brewer  has  in  view,  not 
merely  a  complete  transformation  of  starch  into  sugar; 
indeed,  he  often  wishes  to  prevent  complete  saccharification. 
In  fact,  it  is  especially  important  to  him  to  succeed  in  a  par- 
ticular decomposition  of  starch  and  to  obtain  certain  dex- 
trins  which  resist  the  action  of  yeasts.  He  often  desires  the 
production  of  difficultly  fermentable  sugars  which  remain  in- 
tact during  the  principal  fermentation  and  come  into  play 
only  in  the  after  fermentation. 

The  method  of  decomposition  of  the  starch  influences  to  a 
great  degree  the  character  of  the  beer,  and,  according  to  the 


ROLE   OF  AMYLASE  IN    THE   BRELVERY. 


157 


type  of  beer  that  the  brewer  proposes  to  produce,  more  or 
less  dextrins  and  easily  fermentable  sugars  must  be  formed. 

Under  these  conditions,  the  presence  of  a  large  quantity 
of  diastase  is  undesirable  rather  than  useful.  For  this  reason 
the  brewer,  even  before  he  could  have  known  the  scientific 
reasons,  always  sought  conditions  which  hinder  saccharifica- 
tion  and  the  action  of  an  excess  of  amylase.  Thus  in  drying, 
the  formation  of  dextrins  is  favored,  and  by  saccharification 
at  a  high  temperature  the  excess  of  enzymes  is  destroyed. 

The  influence  of  the  temperature  of  saccharification  on 
the  quantities  of  maltose  and  dextrins  formed  is  shown  in  the 
following  table  from  Petit,  which  shows  the  quantities  of  mal- 
tose and  of  dextrins  formed  at  different  temperatures  as 
well  as  the  relation  between  these  quantities. 


Temperature  of 
Saccharification. 


6o-6l° 
65-66. 
68-69. 
72-73- 


Maltose. 


72 

71-4 
44-7 
24.7 


Ratio. 


30 

1  10.4 

31.8 

1  -.0.44 

57 

1  :  1.27 

76-3 

1  :  3 

We  have  said  above  that  the  methods  of  saccharification 
and  of  drying  influence,  not  only  the  quantity  and  nature  of 
the  dextrins,  but  also  the  nature  of  the  sugar. 

In  fact,  by  saccharifying  starch  under  certain  condi- 
tions, combinations  of  maltose  and  dextrins  are  procured 
which  act  differently  from  maltose  and  dextrins  alone. 

Thus,  when  a  beer-wort  is  left  to  the  action  of  yeast,  it  is 
found  that  the  liquid  still  contains,  when  fermentation  is  fin- 
ished, a  certain  quantity  of  maltose.  The  non-fermentation 
of  the  remaining  sugar  is  not  at  all  due  to  the  exhaustion  of 
the  yeasts,  as  one  might  think  at  first.  Hence  it  is  that  the 
addition  of  pure  maltose  to  the  fermented  wort  induces  a  new 
fermenation  which  exhausts  the  added  sugar,  while  the  sugar 
remaining  in  the  wort  is  hardly  attacked  by  the  yeasts  during 
the  new  fermentation. 


158  THE  ENZYMES  AND   THEIR  APPLICATIONS. 

To  explain  this  fact,  it  is  supposed  that  maltose  can  form 
combinations  with  dextrins,  which  are  called  malto-dextrins. 
These  bodies  have  not  been  isolated  in  a  pure  state  and  their 
chemical  individuality  is  far  from  being  demonstrated.  Yet 
it  is  beyond  doubt  that  a  considerable  difference  exists  in  the 
fermentability  of  the  various  sugars  obtained  by  saccharify- 
ing starch  by  malt  under  different  conditions. 

This  difference  may  be  attributed  either  to  the  actual 
existence  of  different  maltoses  having  different  geometrical 
structures,  or  to  the  formation  of  more  or  less  stable  com- 
binations of  maltose  and  dextrins. 

The  authors  who  have  studied  especially  the  decomposi- 
tion of  starch  by  malt  generally  assume  the  existence  of  differ- 
ent types  of  malto-dextrins  which  are  characterized  by  the  rel- 
ative quantities  of  maltose  and  dextrins  which  they  contain. 

Malto-dextrins  containing  a  great  quantity  of  maltose 
are  called  malto-dextrins  of  a  low  type;  while  malto-dex- 
trins containing  dextrins  in  large  quantity  and  little  maltose 
are  of  a  high  type. 

The  dextrin  contained  in  malto-dextrins  is  transformed 
by  diastase  at  temperatures  higher  than  550,  while  above  630 
malto-dextrins  remain  unattacked.  Beer  yeast  decomposes 
these  combinations  into  fermentable  materials  and  dextrins. 
This  decomposition  is  always  produced  more  or  less  slowly 
according  as  the  yeast  acts  on  a  low  or  high  type  of  malto- 
dextrin. 

The  formation  of  combinations  of  maltose  and  dextrins 
depends  on  the  temperature  of  saccharification.  By  the  ac- 
tion of  the  diastase  below  500  maltose  and  free  dextrins  are 
formed  without  malto-dextrins.  By  allowing  the  diastase 
to  act  between  550  and  620,  the  appearance  of  maltose  com- 
bined with  dextrins  is  observed  and  the  malto-dextrins  in- 
crease considerably  when  this  temperature  is  exceeded.  The 
composition  of  wort,  from  the  point  of  view  of  its  amount  of 
maltose  combined  with  dextrins,  may  consequently  be  regu- 
lated by  the  choice  of  the  temperature  of  saccharification. 


ROLE   OF  AMYLASE  IN    THE  BREIVERY.  159 

According  to  Petit,  there  is  obtained,  with  the  same  malt 
successively  saccharified  at  6o°,  650,  and  69°,  the  following" 
respective  quantities  of  malto-dextrins : 

Temperature 6o°         65°         69° 

Malto-dextrins 2.4%      6.6%       16.2% 

Temperature,  while  influencing  the  formation  of  malto- 
dextrins,  does  not  greatly  influence  the  kind  of  malto-dex- 
trins transformed. 

Thus  the  temperatures  comprised  between  6o°  and  65 ° 
all  produce  the  same  type,  and  it  is  only  at  a  temperature  of 
690  that  one  succeeds  in  appreciably  increasing  the  amount 
of  dextrin  in  the  malto-dextrins  formed. 

The  temperature  of  malting  also  has  a  manifest  influence 
on  the  course  of  the  hydration  of  the  starch. 

Brown  and  Morris,  by  analyzing  worts  obtained  with  four 
malts  prepared  at  different  temperatures  in  ascending  series, 
have  found  the  following  figures. 


Experiments. 

I 

2 

3 

4 

47 

45 

34 

17 

4-25 

7-9 

14.9 

22.4 

1  :o.5 

1  :  1.5 

1  :  2 

1  :  2 

Diastatic  power 

Per  cent  of  malto-dextrins 

Type  of  malto-dextrins  obtained  ... 

As  is  seen,  the  temperature  of  drying  acts  both  on  the 
quantity  and  the  nature  of  the  malto-dextrins.  The  malt 
containing  the  least  diastase  furnishes  both  the  maximum  of 
combined  maltose  and  the  highest  type  of  malto-dextrin. 
The  qualities  and  the  properties  of  the  beer  are  influenced  to 
a  great  degree  by  the  quantity  and  the  type  of  malto-dextrins 
formed  during  manufacture.  These  substances  exert  an  in- 
fluence on  the  attenuation  and  the  taste,  as  well  as  the  pres- 
ervation of  the  beer. 

We  cannot  in  the  present  volume  describe  the  different 
methods  of  brewing  and  we  prefer  to  refer  the  reader  to 
special  works.  Let  us  only  remark  that  by  modifying  the 
mariner  of  hydration  of  the  starch,  beers  of  different  kinds 


160  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

are  produced.  In  fact,  the  method  of  conducting  the  brew- 
ing influences  to  a  great  degree  the  composition  of  the  wort 
which  in  its  turn  acts  on  the  quality  and  type  of  the  beer. 

Even  before  the  decomposition  of  starch  had  been  ex- 
plained theoretically  brewers  understood  the  conditions 
necessary  to  procure  a  wort  having  the  qualities  required  in 
each  case.  When  the  brewer  proposed  to  make  beers  of 
great  attenuation  and  rich  in  alcohol,  he  found  it  necessary 
to  effect  the  brewing  in  such  a  way  as  to  avoid  the  formation 
of  great  quantities  of  malto-dextrins.  When  it  was  a  ques- 
tion, on  the  contrary,  of  a  beer  of  low  fermentation  followed 
by  a  prolonged  secondary  fermentation,  he  sought  to  obtain 
a  great  quantity  of  malto-dextrins  of  a  very  high  type. 

For  top-fermentation  beers  the  manner  of  conducting 
saccharification  also  depends  upon  the  degree  of  density  of 
the  wort.  Wort  intended  for  the  manufacture  of  light  beers 
is  generally  completely  saccharified,  while  for  strong  beers,, 
on  the  contrary,  it  is  sought  to  produce  dextrins  in  much 
larger  proportion. 

It  is  then  by  drying  at  a  suitable  temperature  and  by  the 
duration  of  saccharification  that  one  succeeds  in  producing 
worts  of  .very  different  compositions,  while  using  the  same 
primary  materials. 

BIBLIOGRAPHY. 

Carl  Lintner. — Lehrbuch  der  Bierbrauerei.  Verlag  von  Friedrich  Wieweg 

und  Sohn,  Braunschweig. 
P.  Petit. — La  biere  et  l'industrie  de  la  brasserie.     Paris,  1896. 
Wilhelm  Windisch. — Das  chemische  Laboratorium  des  Brauers.     Berlin. 

Paul  Parey. 
Paul  Lindner. — Mikroskopische  Betriebskontroll  in  den  Garungsgewer- 

ben.     Paul   Parey,  Berlin. 


CHAPTER  XIII. 
MANUFACTURE  OF  MALTOSE. 

By  the  action  of  malt  on  starch  one  can  obtain  accord- 
ing to  the  duration  of  the  process  and  the  temperature  which 
is  employed,  a  series  of  products  differing  in  the  degree  of 
hydration.  By  saccharifying  a  paste  containing  from  5  to  7 
per  cent  of  starch  with  an  infusion  of  malt  at  a  temperature 
of  40°-45°,  an  almost  complete  transformation  of  starch  into 
maltose  is  obtained  after  12  to  15  hours.  The  saccharin 
liquid,  evaporated  to  the  consistency  of  40°-42°  Baume, 
forms  a  white  crystalline  mass  containing  only  1  to  2  parts  of 
dextrin  to  100  parts  of  sugar.  A  product  of  an  entirely  dif- 
ferent nature  is  obtained  by  saccharification  at  6o°-62°.  If 
the  duration  of  saccharification  is  limited  to  30  or  60  minutes 
and  the  work  is  performed  with  an  excess  of  diastase,  a 
strongly  saccharified  syrup  is  obtained  containing  from  20  to 
25  parts  of  dextrin  for  100  of  sugar.  By  saccharification  at 
68°  products  are  obtained  which  possess  only  60  per  cent  of 
maltose. 

These  different  products  owe  their  industrial  applications 
to  the  work  of  Dubrunfaut  and  Cuisenier. 

These  workers  made  a  thorough  study  of  saccharification 
by  malt,  and  they  projected  an  industrial  process  which  ap- 
pears to  have  a  very  great  future  in  store  for  it. 

Dubrunfaut,  in  promoting  the  manufacture  of  maltose, 
hoped  that  the  different  products  of  saccharification  would 
find  many  applications  in  different  industries.  He  believed 
that  pure  maltose  could  replace  with  advantage  cane-sugar 

161 


1 62  THE  ENZYMES  AND    THEIR   APPLICATIONS. 

in  wine-making  and  the  manufacture  of  liqueurs.  The  sac- 
charified syrup  was  to  have  a  place  in  all  the  industries  which 
made  use  of  glucose,  for  example  in  pastry,  in  preparation  of 
preserves,  bon-bons,  etc.  Products  containing  a  large  pro- 
portion of  dextrin  would  be  especially  useful  in  the  brewery 
where  they  would  replace  a  great  part  of  the  malt. 

The  expectations  of  Dubrunfaut  have  not  been  complete- 
ly realized. 

The  maltose  industry  had  a  great  development  at  a  cer- 
tain  period.  Manufactories  were  started  in  France,  Belgium, 
Holland,  and  England,  and  the  production  of  this  sugar 
reached  very  large  proportions.  Of  late  years,  however,  this 
industry,  for  various  reasons,  has  undergone  a  considerable 
falling  off". 

Nevertheless,  the  state  of  the  maltose  industry  does  not 
warrant  the  prediction  that  it  is  destined  to  disappear. 

The  advantages  which  saccharification  by  malt  afford 
over  saccharification  by  acids  are  unquestionable,  and  we  are 
absolutely  certain  that  eventually  this  industry  will  replace 
glucose  manufacture. 

As  the  industrial  preparation  of  maltose  is  very  little 
known,  we  will  give  here  some  information  concerning  its 
technique. 

Potato-flour,  rice-flour  or  maize-flour  is  used  as  the 
source  of  the  product.  From  an  economic  point  of  view 
maize  is  the  raw  material  offering  the  most  advantages.  Un- 
fortunately the  handling  of  this  cereal  presents  great  difficul- 
ties as  to  filtering  and  decoloration  of  the  syrup.  To  pro- 
cure products  of  a  good  appearance  and  to  obtain  satisfac- 
tory yields  it  is  necessary  to  have  strictly  constant  conditions. 

The  successive  operations  are  as  follows : 

ist.  Grinding. 

2nd.   Drying. 

3rd.   Saccharification. 

4th.  Filtration. 

5th.  Clarification. 


MANUFACTURE  OF  MALTOSE.  163 

6th.  Second  filtration. 

7th.  Evaporation. 

8th.  Second  clarification. 

9th.  Evaporation  at  400. 

The  maize,  coarsely  ground,  is  introduced  into  a  horizon- 
tal receptacle  furnished  on  the  inside  with  a  paddle.  Each 
cooker  receives  750  kilograms  of  meal  and  enough  water  to 
give  after  cooking  45  hectolitres  of  liquid.  The  pressure  is 
quickly  raised  while  the  mass  is  agitated,  and  remains  40 
minutes  at  3  atmospheres.  As  it  takes  about  40  minutes  to 
arrive  at  this  pressure,  the  cooking  is  ended  after  about  80 
minutes. 

The  cooked  maize  is  sent  into  a  second  horizontal  recep- 
tacle furnished  with  a  double  wall,  a  Bohm  crusher,  and  a 
paddle.  A  small  amount  of  malt  is  added  at  a  temperature  of 
7°°~7S°>  an<J  in  5  or  10  minutes  liquefaction  of  the  mass 
takes  place.  Then  it  is  cooled  by  the  water  jacket,  the  rest 
of  the  malt  is  added  at  a  temperature  of  65°,  it  is  left  about 
20  minutes  at  this  temperature,  heated  again  to  700,  and  the 
mass  filter-pressed.  For  the  manufacture  of  syrup  contain- 
ing dextrin  saccharification  is  prolonged  for  1  hour  at  68°. 

Malt-sugar  syrup  requires  for  its  manufacture  25  per  cent 
of  fresh  malt.  For  dextrin  syrup  the  quantity  of  malt  is  re- 
duced to  15  per  cent. 

Great  importance  is  attached  to  the  filtering,  and  this 
operation  influences  to  a  great  degree  the  quality  of  the 
product  as  well  as  the  yield. 

The  passage  through  the  filter-press  should  be  made  very 
rapidly,  and  the  filtrate  should  be  perfectly  clear.  An  incom- 
plete filtration  causes  a  change  in  the  juices  and  shows  at  the 
same  time  a  poor  extraction. 

The  slight  turbidity  found  in  badly  filtered  solutions  re- 
veals the  presence  of  a  certain  quantity  of  starch  capable  of 
producing  trouble  during  the  concentration  of  the  juices. 

To  get  a  good  filtration  it  is  essential  to  use  a  malt  whose 


1 64  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

plumule  is  very  long,  and  to  reheat  the  saccharified  wort  at 
a  temperature  of  70 ° '. 

In  maltose  manufactories  filter-presses  of  70  square  cen- 
timetres are  generally  used,  furnished  with  12  frames  cov- 
ered with  linen.  A  battery  of  7  filters  furnishes  in  15  minutes 
45  hectolitres  of  juice  of  2.5 °  to  30  Baume. 

The  filtered  juices  are  placed  in  copper  reservoirs  fur- 
nished with  a  double  wall  for  the  entrance  of  the  steam.  They 
are  reheated  rapidly  to  75  °  and  left  for  about  half  an  hour  at 
this  temperature  for  clarification. 

An  abundant  precipitate  is  formed  which  is  separated  by 
a  second  passage  through  the  filter.  This  second  filtration 
presents  no  difficulties.  It  is  carried  on  in  a  filter-press  of 
small  dimensions. 

The  clear  juices  are  evaporated  in  a  triple-effect  appa- 
ratus where  they  are  concentrated  to  220  Baume. 

The  syrups  are  then  submitted  to  purification  and  to  a 
bone-black  treatment.  The  syrups  are  placed  in  special 
reservoirs  in  which  are  added  10  kilograms  of  powdered 
bone-black  and  500  grams  of  dried  blood  per  25  hectolitres 
of  syrup. 

It  is  kept  boiling  for  10  minutes,  filtered  and  concen- 
trated in  a  vacuum  up  to  40°-42°  Baume.  For  the  manu- 
facture of  products  which  are  highly  clarified  the  syrups, 
after  purification,  are  placed  in  the  battery  of  bone-black  fil- 
ters where  they  remain  from  5  to  8  hours. 

The  yields  usually  obtained  in  the  manufactories  are  from 
92  to  94  kilograms  of  syrup  at  400  per  100  kilograms  of 
maize,  but  to  secure  this  result  a  very  well-conducted  opera- 
tion and  much  attention  are  needed. 

To  give  an  idea  of  the  influence  of  the  method  of  work 
on  the  output  we  may  state  that  in  the  first  years  of  the. 
manufacture  of  maltose  the  yield  was  only  from  60  to  65 
kilograms  of  syrup  per  100  kilograms  of  maize,  and  that  it 
was  only  later,  owing  to  successive  improvements,  that  the 
results  mentioned  above  were  reached. 


MANUFACTURE   OF  MALTOSE.  165 

Properly  prepared  syrup  generally  keeps  well,  but  better 
in  the  open  air  than  in  closed  reservoirs.  In  large  reser- 
voirs exposed  to  the  air,  change  is  never  found,  while  syrup 
placed  in  barrels  often  ferments.  The  analysis  of  the  indus- 
trial products  is  given  below : 

LUMP    MALTOSE. 

Water ^.o. 

Maltose 80.6 

Dextrin 0.2 

WHITE   SYRUP    (FECULA). 

Dry  substances 77. 1 

Maltose 50.2 

Dextrin *74 

SACCHARIFIED    MAIZE. 

Water 20.2 

Maltose 4c 

Dextrin 33. 

Nitrogenous  matter 2.2 

Mineral  substances 0.91 

DEXTRINATED    SYRUPS. 

Water 20. 

Maltose 30.2 

Dextrin 48. 

Nitrogenous  matter 2. 1 

Mineral  substances 0.91 

SYRUPS    OF    RICE. 

Water !8.8 

Maltose yi 

Dextrin 2.4 

Foreign  substances 8.2 


1 66  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

The  maltose  syrup  affords  very  great  advantages  over 
that  of  glucose  in  its  purity  and  economy. 

Maltose  is  a  nutritive  substance  of  great  value.  In  the 
living  organism  it  is  transformed  into  assimilable  sugar  more 
rapidly  than  is  saccharose.  It  is  very  easy  to  digest  and, 
having  not  so  sweet  a  taste  as  cane-sugar,  it  can  be  taken  in 
much  greater  quantities  than  the  latter. 

By  the  action  of  acids  on  starch  industrial  glucoses  are  ob- 
tained which  contain,  besides  dextrins,  foreign  bodies  formed 
under  the  influence  of  the  acids  at  high  temperature.  These 
bodies  give  a  disagreeable  taste  to  glucoses  and  often  possess 
poisonous  properties. 

The  dextrins  formed  under  the  influence  of  acids  have  a 
scant  nutritive  value.  The  pancreatic  juice  acts  very  slowly 
on  these  dextrins  and  its  action  is  always  incomplete. 

As  shown  by  the  experiments  made  by  Soxhlet  and  Stut- 
zer,  dextrins  formed  by  malt  act  quite  differently;  they  are 
much  more  easily  transformed  by  diastases. 

Saccharification  by  malt  affords  another  great  advantage: 
that  of  being  able  to  utilize  amylaceous  materials  directly 
without  going  through  the  manufacture  of  starch. 

By  treating  maize  with  acid  great  modifications  are 
caused  in  the  nitrogenous  matters  as  well  as  in  the  fatty  mat- 
ters. The  products  obtained  are  black,  of  a  disagreeable 
taste,  and  not  suitable  for  the  manufacture  of  beer. 

To  obtain  the  purest  products  it  is  necessary  first  to 
extract  the  starch,  which  entails  great  losses.  Out  of  60 
kilograms  of  starch,  contained  in  100  kilograms  of  maize, 
only  50  to  52  kilograms  are  recovered  in  practice.  One  loses, 
therefore,  8  to  10  kilograms  of  starch,  as  well  as  other  nutri- 
tive substances,  organic  and  mineral,  which  enter  into  the 
composition  of  the  grain  and  which  are  utilized  in  the  manu- 
facture of  maltose. 

The  maltose  industry  also  furnishes  a  more  wholesome 
and  more  nutritive  malt  than  that  furnished  by  the  glucose 
industry.      It  is,  therefore,  indisputable  from  a  hygienic  as 


MANUFACTURE   OF  MALTOSE.  167 

well  as  economic  point  of  view  that  maltose  is  preferable  to 
glucose. 

The  crisis  through  which  the  maltose  industry  is  at  pres- 
ent passing  is  not  likely  to  lead  to  its  abandonment.  This 
manufacture  presents  certain  advantages  and  the  efforts 
made  by  Dubrunfaut  and  Cuisenier  will  not  have  been  in 
vain. 

The  patents  which  protected  this  industry  have  fallen 
into  disuse  and  this  circumstance  will  certainly  not  fail  to 
give  it  a  new  impetus. 


CHAPTER  XIV. 

PANARY   FERMENTATION. 

Dumas'  theory  of  panary  fermentation. — Cerealin  of  Mege-Mouries: — 
The  part  played  by  bacteria  in  panary  fermentation. — The  origin 
of  the  sugar  in  flour. 

The  work  of  bread-making  is  done  in  three  successive 
stages :  kneading,  fermentation  (raising),  and  cooking. 

The  first  of  these  operations  has  for  its  aim  to  make  with 
the  flour  an  elastic  and  homogeneous  dough. 

To  this  end  a  little  yeast  is  diluted  in  warm  water,  flour  is 
added  little  by  little,  then  the  mixture  is  stirred  and  the  mass 
is  kneaded.  Thus  a  dough  is  formed  into  which  a  certain 
quantity  of  salty  water  is  uniformly  worked.  The  kneading 
finished,  the  mass  is  left  for  some  time. 

The  incorporated  yeast  then  causes  a  fermentation  which 
modifies  the  structure  and  the  chemical  composition  of  the 
dough. 

This  fermentation  constitutes  the  second  period,  which  is 
called  "  raising."  It  takes  place  in  the  kneading-trough  and 
generally  lasts  from  20  to  30  minutes. 

The  dough  is  then  divided  into  parts  of  a  certain  size  to 
which  is  given  the  form  of  a  loaf  of  bread.  They  are 
sprinkled  with  flour  and  again  left  quietly  for  30  to  40 
minutes,  after  which  they  are  baked  in  ovens  brought  tip  to 
2500  or  3000. 

The  leaven  used  in  the  preparation  of  bread  comes  from 
a  previous  operation.  After  kneading  the  baker  takes  away 
a  small  quantity  of  the  dough  and  uses  it  as  leaven  in  the 

168 


PANARY   FERMENTATION.  169 

next  operation.  The  same  ferment  is  used  in  this  way  for  an 
indefinite  number  of  times. 

The  principal  agent  in  panary  fermentation  is  a  Sac- 
charomyces.  But  this  is  not  the  only  factor;  others  come  in 
play,  and  here,  too,  are  found  diastatic  actions. 

Corn,  rye,  and  all  other  cereals,  contain  considerable 
quantities  of  amylase  and  substances  which  accelerate  dia- 
static action.  By  grinding,  it  is  true,  a  great  part  of  the  dia- 
stase is  eliminated  with  the  bran,  but  the  flour  is  not  com- 
pletely deprived  of  active  substances.  These  remaining  en- 
zymes play  successively  an  important  part  in  the  various 
stages  in  the  making  of  bread.  The  action  of  the  diastases  of 
the  grains  begins  during  the  milling.  This  action  is  con- 
tinued during  panary  fermentation,  and  may  even  be  evident 
during  baking.  The  part  played  by  the  yeast,  as  well  as  the 
physical  and  chemical  phenomena  which  are  manifested  dur- 
ing bread-making,  have  given  rise  to  different  theories. 

Dumas  regards  panary  fermentation  as  an  alcoholic  fer- 
mentation. According  to  him,  the  starch  and  gluten  of  the 
flour  have  been  already  partially  hydrated  as  a  result  of  mix- 
ing with  water.  This  hydration  would  also  be  favored  by 
the  kneading  which  scatters  the  yeast  uniformly  throughout 
the  mass  and  brings  it  into  contact  with  the  air,  a  condition 
which  favors  fermentation. 

During  the  raising  the  carbonic  acid  formed  in  the 
mass  is  imprisoned  in  the  cavities  of  the  dough,  to  which  the 
gluten  has  given  coherence.  During  baking  the  sudden  ele- 
vation of  temperature  expands  the  gases  enclosed  in  the 
dough,  and  produces  a  swelling  of  the  mass  as  well  as  a  closer 
adherence  among  the  hydrated  materials,  the  starch,  gluten, 
and  albumen. 

According  to  Dumas,  the  carbonic  acid  produced  by 
panary  fermentation  remains  almost  entirely  in  the  bread,  of 
which  it  occupies  about  half  the  volume,  at  a  temperature  of 
ioo°. 

The  yeast  then,  according  to  him,  would  act  by  the  car- 


T7°  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

bonic  acid  it  produces  and  the  fermentation  would  occur  at 
the  expense  of  the  sugar  already  existing  in  the  flour. 

The  theory  of  Dumas  likening  panary  fermentation  to  an 
alcoholic  fermentation  has  met  with  various  objections;  cer- 
tain authors  have  objected  that,  in  panary  fermentation, 
there  is  neither  production  of  alcohol  nor  multiplication  of 
yeasts. 

According  to  Mege-Mouries,  the  bran  contains  an  active 
substance  which  he  calls  cerealin  and  which  has  the  property 
of  transforming  starch  successively  into  dextrin,  glucose,  and 
lactic  acid.  This  substance  is  not  met  with  in  the  flour,  but, 
according  to  Mege-Mouries,  the  gluten  itself  can  saccharify 
the  starch  and  make  it  ferment. 

The  presence  of  alcohol  in  the  dough  after  raising 
escaped  detection  for  a  very  long  time.  Moreover,  different 
experimenters  have  arrived  at  the  conclusion  that  the  yeasts 
introduced  with  the  leaven  do  not  multiply  during  the  rais- 
ing. 

Relying  on  these  data,  and  on  the  almost  invariable  pres- 
ence of  bacteria  in  the  leaven,  some  bacteriologists  have 
evolved  the  hypothesis  that  it  is  the  bacteria  and  not  the 
yeasts  which  produce  the  fermentation. 

In  1883,  Chicandard  described  the  Bacillus  glutinis  which 
lie  considers  is  the  agent  of  panary  fermentation. 

Laurent,  in  his  later  works,  has  described  the  Bacillus 
paniUcans. 

Popoff  has  isolated  from  baker's  dough  an  anaerobic 
bacillus  which,  in  the  presence  of  sugar,  produces  carbonic 
acid  and  lactic  acid. 

The  bacteriological  analyses  of  leavens  made  by  Peters 
and  Boutroux  have  demonstrated  the  constant  presence  in 
the  leaven  of  bacteria  secreting  diastase  and  acting  on  starch 
and  albuminoid  matters.  Furthermore,  the  presence  of  bac- 
teria of  the  same  nature  in  corn-meal  has  been  ascertained. 

The  constant  intervention  of  ferments  in  bread-making 
may  then  be  considered  as  demonstrated. 


PANARY   FERMENTATION.  ill 

According  to  some,  the  bacteria  alone  cause  fermenta- 
tion; according  to  others,  the  bacteria  act  in  symbiosis  with 
the  yeast :  the  former,  by  the  aid  of  their  diastase,  would 
furnish  sugar  to  the  yeasts. 

Wolffin  succeeded  in  producing  normal  bread  by  replac- 
ing the  leaven  by  a  culture  of  Bacillus  levans.  Analogous  ex- 
periments have  been  made  by  Popoff  with  the  same  success. 

Boutroux,  who  has  taken  up  these  experiments  again, 
and  carefully  studied  bakery  yeast,  has  reached  the  following 
conclusions : 

ist.  Alcoholic  yeast  is  always  present  in  the  leaven  of 
bread. 

2nd.  This  yeast  is  cultivated  from  dough  to  dough  in 
such  a  way  that  by  sowing  a  first  dough  with  imponderable 
traces  of  yeast,  there  will  be  found,  at  the  end  of  several 
operations,  a  uniform  distribution  of  yeast  in  the  dough. 

3rd.  The  other  micro-organism  found  in  the  dough,  and 
to  which  may  be  hypothetically  attributed  the  power  of 
making  it  rise,  acts  quite  differently :  transferred  from  dough 
to  dough,  it  ceases  to  produce  fermentation  after  the  second 
or  third  operation. 

The  presence  in  the  leaven  of  bacteria  favoring  bread- 
making  is  on  the  whole  an  exceptional  phenomenon. 

It  appears  from  the  studies  of  Boutroux  that  generally 
the  presence  of  bacteria  is  unfavorable:  they  attack  the  glu- 
ten and  prevent  the  bread  from  rising.  In  practical  baking 
the  destructive  action  of  these  bacteria  is  checked  by  the 
presence  of  the  yeast  which,  in  a  normally  constituted  dough, 
finds  an  excellent  field  for  development,  antagonizes  the 
foreign  organisms,  and  is  alone  of  importance  in  panary  fer- 
mentation. 

The  opinion  of  Dumas  is  also  confirmed  by  the  experi- 
ments of  Moussette  and  Aime  Girard,  who  have  succeeded 
in  ascertaining  the  presence  of  alcohol  in  the  products  of 
panary  fermentation. 

Moussette,  by  condensing  the  steam  of  bread-ovens  dur- 


172  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

ing  baking,  has  obtained  an  alcoholic  solution  containing  1.6 
per  cent  of  alcohol. 

According  to  Girard,  the  same  weight  of  alcohol  as  of 
carbonic  acid  is  formed  during  fermentation.  He  finds 
nearly  2.5  grams  of  each  of  these  substances  per  kilogram  of 
bread. 

According  to  some  authors,  the  sugar  consumed  in  pan- 
ary  fermentation,  and  which  equals  nearly  1  per  cent  of  the. 
weight  of  the  flour,  comes  directly  from  the  grain. 

To  support  this  opinion  we  may  mention  barley,  which, 
always  contains  appreciable  quantities  of  fermentable  sugars. 
But  the  amount  of  sugar  in  corn  is  really  very  variable, 
though  it  is  observed  that  cereals  which  contain  quantities 
of  fermentable  materials  insufficient  for  panary  fermentation 
nevertheless  ferment  as  energetically  as  cereals  which  are 
rich  in  sugar.  On  the  other  hand,  flour  which  is  deprived  of 
certain  constituent  parts  of  the  grains  is  found,  as  a  result, 
poorer  in  sugars. 

According  to  Aime  Girard,  Boutroux,  and  Morris,  there 
is  produced  during  the  growth  of  gramineous  plants  an  ac- 
cumulation of  sugar  in  the  stem;  this  sugar,  at  the  time  of 
the  formation  of  starch,  passes  into  the  embryo  of  the  grains 
and  is  there  transformed  into  starch  as  the  grain  ripens. 

As  a  result,  there  will  be  found  only  traces  of  sugar  in 
the  ripe  corn,  and  the  flour  will  be  free  from  natural  sugars 
since  during  grinding  the  greater  part  of  the  germ  is  carried 
away. 

In  view  of  this  fact,  it  is  pertinent  to  ask  whence  comes 
the  sugar  which  serves  for  fermentation.  According  to 
Poehl,  the  fermentable  sugar  found  in  flour  is  produced  dur- 
ing the  grinding  of  the  grains,  as  the  result  of  a  diastatic  ac- 
tion on  the  starch.  This  diastatic  action  is  manifested  only 
with  the  grains  containing  a  certain  quantity  of  water,  while 
the  dry  grains  do  not  furnish  any. 

Thus,  when  one  treats  a  grist  of  corn  containing  11  to 
13  per  cent  of  water  with  900  alcohol,  one  finds  in  the  liquid 


PA  NARY  FERMENTATION.  173 

the  presence  of  reducing  sugar.  The  same  grain,  previously 
dried  and  then  submitted  to  the  same  treatment  by  alcohol, 
furnishes  no  sugar. 

There  is  really  then  a  transformation  of  starch  into  sugar 
and  the  action  of  amylase  is  consequently  shown  at  the  time 
of  grinding.  It  is  quite  reasonable  to  suppose  that  hydra- 
tion once  begun  continues  during  kneading  and  raising, 
although  the  amount  of  sugar  does  not  perceptibly  increase 
during  these  stages  of  the  work. 

The  intervention  of  diastase  is  shown  with  more  clearness 
during  baking.  The  dough,  once  introduced  into  the  oven, 
heats  very  unequally.  At  the  surface  the  temperature  rises 
abruptly  and  causes  the  formation  of  a  crust  which  prevents 
the  volatilization  of  the  gases  and  the  water  vapor  formed. 
Inside  the  temperature  rises  very  slowly,  a  circumstance 
which  favors  alcoholic  fermentation  as  well  as  diastatic  ac- 
tion, then  the  diastases  continue  to  act  up  to  a  temperature 
of  8o°.  Under  the  action  of  the  water  vapor  and  the  heat, 
the  grains  of  starch  are  transformed  into  soluble  starch  and 
amylo-dextrins. 

The  small  quantity  of  diastase  contained  in  the  flour  is  in 
excellent  condition  to  cause  hydration  of  the  starch  paste, 
which  cannot  be  formed  except  in  very  small  quantity,  owing 
to  the  lack  of  water.  It  is  especially  during  baking  that  mal- 
tose and  dextrins  are  formed  in  the  bread  and  give  to  it  a 
characteristic  taste  and  consistency.  Flour  of  superior  qual- 
ity generally  contains  small  quantities  of  diastase,  while 
flours  containing  a  certain  quantity  of  bran  are  richer  in  ac- 
tive materials  which  influence  to  a  great  degree  the  character 
of  the  bread.  Thus  the  soft  crumb  of  brown  bread  is  due  ex- 
clusively to  the  diastase  of  the  bran. 

White  bread,  soaked  in  warm  water,  furnishes  a  half-solid 
mass  and  only  about  6  per  cent  of  the  materials  dissolve. 
Brown  bread,  treated  in  the  same  way,  gives  to  the  water  a 
milky  aspect  and  45  to  50  per  cent  of  the  dry  matter  is  dis- 
solved.    This  difference  in  solubility  comes  from  the  differ- 


174  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

ence  between  the  modes  of  action  of  the  diastase  in  the  two 
kinds  of  bread. 

In  bran,  in  the  germs  of  corn,  and  consequently  in  the 
flour  too,  there  are  still  other  enzymes  which  take  part  in  the 
bread-making. 

The  transformation  which  the  gluten  undergoes  during 
the  raising  and  baking  appears  to  us  to  be  due  to  a  dia- 
static  action,  but  this  question  is  not  yet  very  clearly  demon- 
strated. 

The  intervention  of  enzymes  is  much  more  evident  in  the 
coloring  of  flour. 

In  flour,  there  are  found  oxidizing  enzymes  to  which  we 
shall  have  occasion  to  return  in  studying  oxidases. 

BIBLIOGRAPHY. 

Dumas. — Traite  de  chimie  applique  aux  arts.     Paris,   1843. 

Birnbaum. — Das  Brotbacken. 

Leon  Boutroux. — Le  pain  et  la  panification;  chimie  et  technologie  de  la 

boulangerie  et  de  la  meunerie. 
Aime    Girard. — Sur    la   fermentation    panaire.     Comptes    Rendus,    t.    CI, 

p.   605. 
Boutroux. — Contribution  a.  l'etude  de  la  fermentation  panaire.     Comptes 

Rendus,  1883,  p.  116. 
Moussette. — Observations  sur  la  fermentation  panaire.     Comptes  Rendus, 

1865,  XCV. 
Lehman. — Ueber  die  Sauerteiggahrung  und  die  Beziehungen  des  Bacillus 

levans   zum   Bacillus   coli   communis.     Centralbl.    fur    Bakteriologie, 

1894. 
W.  L.  Peters. — Die  Organismen  des  Sauerteigs  und  ihre  Bedeutung  fiir 

die  Brotgahrung.     Botanische  Zeitung,  1889. 


CHAPTER  XV. 

ROLE  OF  AMYLASE  IN  THE  DISTILLERY. 

Treatment  of  grains  by  acid  and  by  malt.— Influence  of  heating  on  sac- 
charification. — Choice  of  temperature  of  saccharification  —  Principal 
and  secondary  saccharification.— Experiments  of  Effront  on  change 
in  diastases  during  saccharification.— The  infusion  process.— Change 
in  diastases  during  the  successive  stages  of  the  work.— Control  of  the 
work  in  the  distillery. 

Amylaceous  materials  do  not  ferment  directly  by  the  ac- 
tion of  yeast.  To  make  them  readily  subject  to  the  alcoholic 
fermentation,  it  is  necessary  to  submit  them  to  a  previous 
saccharification. 

To  produce  this  transformation,  the  distiller  has  for  a 
long  time  used  mineral  acids,  and  it  is  only  in  recent  years 
that  these  agents  have  almost  wholly  disappeared  from 
manufactories,  where  they  have  been  replaced  by  malt. 

The  use  of  acids  as  saccharifying  agents  presents,  as  a 
matter  of  fact,  some  notable  disadvantages.  To  obtain  a 
complete  saccharification  without  considerable  loss  of  the 
sugar  formed,  it  is  necessary  to  have  very  dilute  mashes,  to 
keep  them  for  a  very  long  time  at  a  temperature  in  the  neigh- 
borhood of  ioo°,  and  to  use  considerable  quantities  of  acid 
which  must  necessarily  be  neutralized  before  the  addition  of 
the  yeast.  Saccharification  by  acids  is,  therefore,  not  very 
economical,  moreover  it  is  never  complete,  and  the  greatest 
yields  which  can  be  obtained  are  never  above  50  or  53  litres 
of  alcohol  per  100  kilograms  of  starch  used. 

Working  with  acid  presents  still  another  disadvantage: 
it  gives  a  residue  which  cannot  be  utilized  for  food  for  cattle, 
a  disadvantage  sufficient  to  condemn  the  method. 

175 


I?6  THE  ENZYMES  AND    THEIR   APPLICATIONS. 

By  using  malt  all  the  disadvantages  of  the  acid  process 
disappear,  and  saccharification  takes  place  comparatively 
rapidly.  The  malts  obtained  in  this  way  are  of  good  quality 
and  the  yield  in  alcohol  exceeds  65  litres  for  each  100  kilo- 
grams of  starch  used.  Nevertheless,  the  malt  process  has 
its  difficulties,  also.  It  is  not  always  easy  to  prepare  a  malt 
corresponding  to  the  needs  of  the  distillery,  and  it  is  often 
very  difficult  to  use  it  to  the  best  advantage. 

Of  all  the  industries  which  use  diastase  as  a  saccharifying 
agent,  the  distillery  undoubtedly  has  the  most  difficulties  to 
contend  with  in  the  use  of  amylase.  It  is,  in  fact,  the  amy- 
lase which  plays  the  principal  part  in  this  industry,  for  it  reg- 
ulates the  course  of  fermentation,  and  influences  all  the 
stages  of  the  work. 

A  thorough  knowledge  of  the  method  and  conditions  of 
the  action  of  this  diastase  is,  therefore,  indispensable  in  order 
to  direct  the  work  suitably. 

For  this  reason,  in  studying  the  process  of  distillation 
solely  from  the  point  of  view  of  the  part  played  by  the  malt, 
we  may  review  the  successive  operations  in  that  industry. 

Cooking. — Starch  removed  from  the  cells  is  not  easily 
attacked  by  amylase  but,  when  it  is  not  freed  from  the 
grains  which  enclose  it,  its  transformation  by  diastase  is  still 
more  difficult.  The  intercellular  substances  and  the  cellu- 
lose membrane  of  starch-containing  cells  prevents  contact  of 
the  enzyme  with  the  granules  of  starch. 

To  render  efficacious  the  action  of  the  diastase  on  amy- 
laceous materials,  it  is  necessary  to  submit  them  to  a  cook- 
ing which  dissolves  the  intercellular  substances  and  frees  the 
grains  of  starch. 

By  working  with  finely  ground  amylaceous  materials,  the 
combined  actions  of  heat  and  water  favor  to  a  great  degree 
the  action  on  the  starch,  and  cooking  in  contact  with  the 
air  is  sufficient  to  obtain  a  paste  which  is  easily  saccharified 
by  amylase.  However,  in  working  with  whole  grains,  it  is 
necessary  to  work  under  pressure. 


ROLE   OF  AMYLASE  IN    THE  DISTILLERY.  177 

In  practice,  the  steaming  is  done  in  closed  vessels,  where 
the  grains  are  submitted  for  about  2  hours  to  a  pressure  of  3 
to  4  atmospheres. 

Increase  in  temperature  is  very  favorable  to  the  solution 
of  the  starch,  but  it  presents  great  disadvantages  from  other 
points  of  view. 

The  principal  part  of  the  grains,  the  starch,  resists  high 
temperatures  without  decomposing,  but  this  is  not  true  of 
the  other  substances  constituting  the  grain,  of  the  sugars, 
for  example,  which  are  destroyed  at  high  temperature.  By 
cooking  a  mash  containing  sugar  at  different  temperatures, 
it  is  found  that  the  destruction  of  the  sugar  increases  in  a 
great  degree  in  proportion  as  the  pressure  increases. 

Thus  a  mash  containing  15  per  cent  of  maltose  kept  for 

£  hour  at  2  atmospheres  loses  0.85  of  sugar. 

«       "  3  "  1.7 

"      "  4  "  3-4 

Grains,  and  especially  potatoes,  contain  quite  consider- 
able quantities  of  fermentable  sugars,  and  the  destruction  of 
these  must  necessarily  bring  about  a  perceptible  loss  in  al- 
cohol. 

High  pressure  also  has  the  effect  of  dissolving  different 
substances  which  enter  into  the  composition  of  the  grains. 
The  increase,  in  the  mash,  of  the  quantity  of  extractive 
substances  under  the  influence  of  high  pressures  is  consid- 
ered by  different  authors  as  a  proof  of  the  efficiency  of  steam- 
ing. It  is  on  this  basis  that  we  are  sometimes  advised  to  ex- 
ceed the  pressure  of  3  atmospheres  during  cooking.  It  is 
unquestionable  that  high  pressure  increases  the  density  of 
the  mash,  and  that  it  favors  increase  in  the  quantity  of  reduc- 
ing substances,  but  this  fact  does  not  necessarily  mean  an 
increase  in  the  alcoholic  output.  On  the  contrary,  numer- 
ous experiments  made  to  this  end  have  shown  that  a  mash  of 
much  cooked  grains,  while  giving  a  good  saccharification 
with  amylase,  furnishes  a  yield  in  alcohol  inferior  to  that  of 


1 78  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

a  mash  prepared  at  a  moderate  pressure.  Thus  three  grain 
mashes,  prepared  at  different  pressures,  but  with  other  con- 
ditions constant,  give  the  following  results : 

Density         Alrnhnl         Diastatic 
Balling.        Alconoi.         Powen 

2  atmospheres 17  10.5  40 

3  "  18.1       10.3  28 

4  " 18.6        9.8  13 

It  is  seen  that  the  mash  cooked  at  4  atmospheres  pos- 
sesses a  density  of  18.6,  while  the  mash  cooked  at  two  atmos- 
pheres shows  a  density  of  only  17.  We  may  observe  at  the 
same  time  that  the  maximum  amount  of  sugar  does  not  cor- 
respond to  the  greatest  yield  of  alcohol.  Indeed  the  must 
prepared  at  2  atmospheres  furnishes  10.5%  of  alcohol,  while 
the  must  prepared  at  4  atmospheres  gives  only  9.8%.  Un- 
der the  heading  "  Diastatic  Power  "  we  find  an  explanation 
of  this  anomaly. 

The  grains  cooked  at  2  atmospheres  and  then  sacchari- 
fied under  the  same  conditions  show  a  fermenting  power  of 
40;  the  diastatic  power  diminishes  with  the  increase  of  pres- 
sure and  at  4  atmospheres  a  fermenting  power  of  only  13  is 
found.  The  cooking  of  the  must  gives  rise  to  certain  sub- 
stances which  weaken  the  enzymes  during  saccharification. 
The  cooking  under  high  pressure  therefore  brings  about,  as 
immediate  consequence,  an  incomplete  fermentation.  The 
nature  of  the  harmful  substances  is  not  exactly  known,  neither 
can  it  be  determined  what  are  the  bodies  which  give  rise  to 
them ;  nevertheless  the  formation  of  substances  impeding 
diastatic  action  cannot  be  doubted.  It  is  well  to  take  note 
of  this  fact,  especially  when  it  is  proposed  to  work  with  a 
limited  quantity  of  diastase. 

The  most  rational  method  of  wrork  consists  in  making  a 
very  fine  meal  of  the  grains,  and  in  cooking  this  meal  for  1^ 
to  2  hours  with  water  at  i|  or  2  atmospheres  at  the  most. 

Under  these  conditions,  mashes  are  obtained  which  do 
not  weaken  the  diastase.     This  mode  of  work  also  offers  the 


ROLE   OF  AMYLASE  IN    THE  DISTILLERY.  179 

great  advantage  of  furnishing  a  much  more  wholesome  malt 
than  that  obtained  by  cooking  at  high  pressure. 

It  is  very  difficult  to  show  in  a  conclusive  way  the  un- 
favorable influence  which  cooking  at  high  pressure  has  upon 
the  quality  of  the  malts.  Chemical  analysis  gives  us  data  on 
the  amount  of  nitrogen,  phosphates,  and  organic  materials, 
in  the  malts,  but  it  does  not  give  us  any  information  as  to 
their  nutritive  value,  and  the  comparative  value  of  the  malts 
cannot  be  determined  except  by  experiments  on  animals. 

Experiments  of  this  kind  would  have  to  be  made  in  an 
agricultural  station  having  a  distillery  at  its  disposal.  We 
do  not  think  that  experiments  of  this  kind  have  been  at- 
tempted, and  at  any  rate  we  do  not  know  the  results  which 
they  have  given.  Still,  our  opinion  on  the  comparative 
value  of  different  malts,  according  to  the  temperatures  at 
which  the  cooking  has  been  done,  results  from  an  inquiry 
we  have  made.  Information  we  have  gained  from  different 
distillers  and  agriculturists  proves  that  cattle  eat  more 
readily  malts  obtained  by  cooking  the  grains  at  slight  pres- 
sure. These  malts  can  be  consumed  by  them  in  greater 
quantities  than  the  malts  cooked  under  high  pressure. 
These  same  malts  have  not,  like  those  prepared  at  high  pres- 
sure, an  unfavorable  effect  on  the  quantity  and  the  quality  of 
the  milk. 

The  influence  of  cooking  on  the  nutritive  value  of  the 
malts  may  be  particularly  observed  in  towns  possessing 
several  distilleries.  The  farmer  who  buys  liquid  malts 
always  ends,  after  longer  or  shorter  trials,  by  giving  prefer- 
ence to  one  of  the  distilleries,  and  this  preference  is  always 
in  favor  of  the  manufactory  using  low  pressure. 

It  appears  to  us  probable  that  the  same  substances  which 
influence  saccharification  unfavorably  hinder  digestion  of  the 
malts  obtained  by  cooking  at  high  pressure. 

Saccharification  of  Amylaceous  Materials. — By  cook- 
ing, the  substances  which  in  the  grains  are  interposed  be- 
tween the  starch-bearing  cells  are  partially  dissolved,  and 


180  THE  ENZYMES  AND   THEIR  APPLICATIONS. 

the  starch-cells  are  liberated  from  the  tissues  where  they 
were  enclosed. 

Inside  the  cells  the  grains  of  starch  first  swell  and  then 
liquefy.  To  remove  this  starch  from  the  cells,  it  is  necessary 
to  have  recourse  to  a  mechanical  action  which  bursts  the 
cellulose  membrane,  which  resists  very  strongly  the  action 
of  heat.  This  bursting  is  necessary  because,  if  the  liquid 
starch  remains  enclosed  in  the  cells,  it  will  undergo  an  in- 
complete saccharification  only. 

To  this  end  the  cooked  mass  is  vigorously  stirred,  the 
mash  is  expelled  from  the  macerater  by  strong  pressure, 
and  the  action  is  completed  by  a  crushing  which  breaks  up 
the  mass  and  bursts  the  most  resistant  cells.  The  mash  thus 
prepared  is  suitably  cooled,  malt  is  added,  and  it  is  left  to 
saccharify. 

The  determination  of  the  temperature  at  which  saccharifi- 
cation should  be  carried  on  has  been  made  both  by  manu- 
facturers and  by  scientists.  Still,  in  spite  of  all  the  efforts  of 
this  kind,  the  question  is  yet  unsettled  on  account  of  the 
divergence  of  opinion  on  the  subject.  To  understand  the 
difficulties  which  are  met  with  in  the  choice  of  the  tempera- 
ture for  saccharification,  one  must  first  of  all  realize  the 
many  and  varied  results  which  it  is  desired  to  obtain  by  this 
operation,  namely:  the  liquefaction  of  the  starch  of  the  raw 
grains  and  the  proper  utilization  of  the  starch  of  the  malt. 

Finally  one  must  take  account  of  the  presence  of  germs 
and  bacteria  in  the  malt,  as  well  as  the  acidity  of  the  medium 
and  the  change  in  the  diastase. 

Theoretically,  by  a  very  prolonged  action  of  the  malt  on 
the  starch,  a  complete  saccharification  is  obtained,  but  in 
practice  it  is  absolutely  impossible  to  procure  a  complete 
transformation  and  the  saccharification,  under  the  best  con- 
ditions, only  furnishes  80  parts  of  maltose  per  100  of  starch. 

The  hydration  of  the  starch  is  accomplished  in  the  work 
of   the    distillery   in   two   different    stages :  saccharification 


ROLE    CF  AMYLASE  IN    THE  DISTILLERY.  i«t 

proper,  then  the  secondary  saccharification,  which  is  pro- 
longed throughout  the  duration  of  fermentation. 

Of  these  two  saccharifications,  the  last  is  the  most  dif- 
ficult to  regulate,  and  it  is  generally  believed  that  it  is  best 
to  produce  the  greatest  part  of  the  sugar  in  the  first  stage 
of  hydration,  so  as  to  leave  the  fewest  possible  dextrins  for 
the  secondary  saccharification.  To  this  end  the  most  favor- 
able conditions  must  be  employed  during  the  principal  sac- 
charification and  a  temperature  must  be  adopted  which 
furnishes  the  greatest  effect  in  the  least  time.  It  is  the 
determining  of  this  temperature  that  offers  the  first  difficul- 
ties. The  optimum  temperature  of  diastases  is  far  from 
being  constant.  In  fact,  if  we  compare  the  quantities  of 
maltose  formed  during  the  same  period  of  time  with  a  given 
quantity  of  malt,  at  different  temperatures,  we  shall  find  that 
the  maximum  of  sugar  formed  takes  place  at  very  different 
temperatures  according  to  the  duration  of  saccharification. 

Thus,  when  different  specimens  of  the  same  starch  are 
saccharified  for  I  hour  with  the  same  quantity  of  malt,  work- 
ing at  temperatures  increasing  from  300  to  700,  an  optimum 
temperature  of  6o°  to  630  is  found. 

When  the  same  experiment  is  repeated  by  prolonging 
the  duration  of  saccharification  for  three  hours,  the  optimum 
temperature  is  reduced  to  500,  and  it  descends  to  300  if 
saccharification  is  made  to  last  for  12  hours. 

From  this  it  results  that  the  selection  of  the  tempera- 
tures of  saccharification  depends  on  the  duration  of  the  latter, 
and  that  the  longer  the  duration  of  the  action,  the  lower 
should  be  the  temperature  of  saccharification. 

In  practice  the  duration  of  saccharification  is  very  varied. 
This  operation  lasts,  in  different  manufactories,  from  20 
minutes  to  2  hours.  Its  duration  is  determined  by  the  kind 
of  plant,  and  by  the  general  conditions  of  the  work. 

If  a  complete  hydration  of  the  starch  is  desired,  it  is 
always  well  to  choose,  when  saccharification  lasts  a  half- 
hour,  a  temperature  of  620  to  630,  while  one  must  lower  the 


182  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

temperature  to  57°-58°  for  saccharifications  lasting  from  i  to 
2  hours. 

We  now  know  the  conditions  necessary,  in  the  principal 
saccharification,  for  a  complete  hydration. 

But  in  reality  the  quantity  of  sugar  formed  during  the 
first  stage  of  saccharification  has  very  little  influence  on  the 
final  result  of  the  operation.  A  dextrinated  mash  affords  as- 
much  alcohol  as  one  strongly  saccharified.'  Moreover,  the 
quantity  of  diastase  indispensable  for  the  secondary  saccha- 
rification is  no  greater  for  the  dextrinated  mash  than  for  the 
mash  which  already  contains  a  large  amount  of  sugar. 

A  very  long  series  of  experiments  performed  in  this  way- 
have  shown  us  that  the  intensity  of  the  first  saccharification 
is  of  little  importance,  and  that  the  final  result  depends 
especially  on  the  more  or  less  complete  preservation  of  the 
diastase  during  fermentation. 

Still,  the  principal  saccharification  cannot  be  completely 
suppressed.  It  has  a  reason  for  existing,  especially  from  the 
point  of  view  of  liquefaction.  In  fact,  it  is  this  first  opera- 
tion which  gives  to  the  cooked  mass  the  necessary  fluidity. 
Moreover,  it  succeeds  in  attacking  the  cells  which  have 
escaped  the  action  of  steam,  and  it  liquefies  the  particles  of 
starch  which  adhere  to  the  spent  malts. 

By  raising  the  temperature  of  saccharification  above  60% 
excellent  conditions  are  obtained  for  liquefaction. 

We  may  now  consider  the  change  in  the  diastase  under 
the  action  of  heat,  for  the  active  substance  which  is  to  serve 
for  the  secondary  saccharification  must,  after  the  principal 
saccharification,  be  absolutely  unchanged.  The  tempera- 
ture of  saccharification  must,  therefore,  necessarily  be  lower 
than  that  at  which  the  diastase  commences  to  weaken.  All 
the  authors  who  have  investigated  saccharification  are  com- 
pletely in  accord  on  this  point,  but  their  opinions  are  very 
different  when  it  is  a  question  of  saying  at  what  temperature 
the   change  begins.     According  to   some,   the  active  sub- 


ROLE   OF  AMYLASE  IN    THE  DISTILLERY.  183 

stance  of  the  malt  can  withstand  temperatures  of  620  with- 
out changing.  According  to  others,  the  degree  of  resist- 
ance of  the  malt  to  high  temperature  depends  on  the  dura- 
tion of  the  action  as  well  as  upon  the  concentration  and  the 
composition  of  the  mash.  According  to  some  chemists,  a 
temperature  of  6o°-62°  would  bring  about  in  diluted  mashes 
a  pronounced  change  of  the  diastase,  while  in  concentrated 
mashes  the  amylase  would  resist  much  better.  Others 
finally,  make  a  notable  difference,  from  the  point  of  view  of 
the  preservation  of  the  enzyme,  between  a  dextrinated  must 
and  a  saccharified  must. 

It  is  assumed  that  the  presence  of  great  quantities  of 
maltose  in  the  solution  protects  the  diastase  against  the 
disastrous  effect  of  high  temperatures  and,  on  that  basis, 
it  would  be  advisable  to  conduct  saccharification  in  two 
stages,  during  the  first  30  minutes  of  the  action  of  the 
malt    at    a    temperature    of    58°-6o°,    then    raising    it    to 

64°-67°. 

For  the  support  of  this  theory  numerous  experiments 
are  cited  which,  however,  do  not  lead  to  very  clear  conclu- 
sions. The  various  determinations,  made  by  several  chem- 
ists with  different  raw  materials,  under  conditions  neces- 
sarily varied,  and  by  diverse  methods,  cannot  furnish  data 
of  sufficient  accuracy  to  solve  this  question. 

The  greater  or  less  change  in  the  diastase  at  different 
temperatures  may  be  demonstrated  by  a  very  simple  method. 

To  a  starch  paste  is  added  a  quantity  of  malt  just  suffi- 
cient to  produce,  under  favorable  conditions,  a  complete 
saccharification.  After  this  addition,  two  samples  are 
taken;  one  is  left  for  12  hours  at  a  temperature  of  30°,  the 
other  is  first  kept  for  an  hour  at  a  high  temperature,  then 
for  1 1  hours  at  300. 

If  under  these  conditions  a  difference  is  found  in  the 
amount  of  maltose,  this  proves  the  influence  of  high  tem- 
peratures. 


1 84  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

Here  are  three  experiments  made  at  different  tempera- 
tures: 

Maltose  formed  : 


After  i  hour.       After  12  hours. 


.  j  12  hours  at  300  C 2.4  9.6 

(    1  hour  at  50°  C.  and  11  hours  at  300  C 8.3  10.2 


R  j  12  hours  at  30°  C 2.2  9.8 

(    1  hour  at  55°  C.  and  n  hours  at  300  C 9.1  11. 6 

P  \  12  hours  at  300  C 2.2  9.9 

•    1  hour  at  59°  C.  and  n  hours  at  300  C 9.5  9.7 

The  maltose,  in  all  these  experiments,  was  measured  after 
1  hour  and  after  12  hours  of  saccharification. 

Mashes  kept  at  450,  500,  and  59°  furnished,  after  the  first 
hour  of  saccharification,  a  quantity  of  sugar  much  greater 
than  that  produced  in  the  sample  specimen  left  at  300.  After 
saccharification  at  590  for  1  hour,  9.5%  of  maltose  is  ob- 
tained, in  place  of  2.2%  obtained  in  the  same  length  of  time 
at  300. 

If  the  diastase  had  not  changed  during  the  first  hour  of 
saccharification  at  590,  at  the  end  of  the  11  subsequent  hours 
a  much  larger  quantity  of  sugar  would  be  obtained  than  in 
the  sample  specimens,  since  in  the  first  hour  of  saccharifica- 
tion it  was  already  much  more  advanced  than  in  the  sample 
specimens.  But  such  was  not  the  case.  After  12  hours  of 
saccharification,  there  was  found  in  the  sample  specimen 
9.9%  of  maltose,  while  in  the  experiment  where  the  diastase 
was  carried  for  an  hour  at  590  there  was  found  only  9.7%  of 
sugar.  The  temperature  of  570  is,  therefore,  the  limit  to 
which  amylase  can  be  carried  for  1  hour  without  producing  a 
noticeable  change. 

The  influence  of  high  temperatures  of  saccharification 
may  be  equally  well  shown  by  the  following  experiments : 

In  different  experiments,  at  different  temperatures, 
digest  a  litre  of  paste  containing  10  grams  of  starch  and  5 
cubic  centimetres  of  malt  infusion. 


ROLE   OF  AMYLASE  IN    THE  DISTILLERY.  185 

Experiment.  Duration  of  Saccharification.  Starch  transformed. 

i        12  hours  at  300 85% 

2  i   hour  at  450    and  1 1  hours  at  300.  97 

3  "  5o° 96 

4  "  640 68 

By  repeating  the  same  experiments  with  mashes  of  dif- 
ferent concentrations  and  containing  different  proportions  of 
dextrins  and  maltose,  we  have  been  able  to  ascertain 
definitely  the  concentration  and  the  amount  of  maltose  exer- 
cising a  protective  action  on  the  diastase,  but  that  this  ac- 
tion is  slight  and  that  it  may  be  entirely  neglected  when  580 
is  exceeded. 

At  temperatures  higher  than  580,  even  when  the  mashes 
are  very  concentrated,  a  great  destruction  of  diastase  occurs. 

By  working,  as  is  the  case  in  most  distilleries,  with  a  great 
quantity  of  malt,  the  lack  of  diastase  in  the  secondary  fer- 
mentation is  not  perceived,  but  the  result  is  quite  different 
when  there  are  rational  conditions  of  work  and  when  one 
seeks  to  reduce  the  quantity  of  malt  to  what  is  strictly  neces- 
sary. 

Those  who  recommend  high  temperatures  of  saccharifi- 
cation bring  other  arguments  to  the  support  of  their  opinion- 
According  to  them,  one  must  employ  a  temperature  of  60  ° 
or  even  a  higher  one,  because  otherwise  the  starch  of  the 
malt  will  not  be  utilized  to  the  best  advantage,  and  because 
only  a  high  temperature  can  weaken  the  bacterial  ferments 
which  are  always  present  in  the  malt. 

The  utilization  of  the  starch  in  malt  involves  great  diffi- 
culties, because  its  complete  saccharification  is  secured  only 
at  a  temperature  of  700. 

At  650   one  finds     4%  of  starch  still  undissolved. 

"   6o°  "  8% 

"  50°  "        73% 

Hence  in  the  choice  of  a  temperature  of  saccharification 


*86  THE  ENZYMES  AND    THEIR   APPLICATIONS. 

one  must  take  into  account  the  starch  of  the  malt.  By 
choosing  a  temperature  of  55 °  one  runs  the  risk  of  losing  42 
per  cent  of  the  starch  contained  in  the  malt,  while  at  a  tem- 
perature of  6o°  the  loss  is  considerably  less.  At  this 
temperature  there  remains  only  8  per  cent  of  amylace- 
ous material  not  attacked.  When  using  from  12  to  16 
per  cent  of  malt  one  is  obliged  to  choose  a  high  temperature 
of  saccharification,  but  when  working  with  a  very  much  re- 
duced quantity,  one  may  choose  a  lower  temperature,  be- 
cause the  loss  of  starch  is  reduced  in  this  case  to  a  minimum. 
Furthermore,  the  losses  in  amylaceous  materials  which  may 
come  as  the  result  of  a  poor  extraction  of  starch  are  never 
-as  detrimental  to  the  yield  as  the  weakening  in  the  diastase 
under  the  influence  of  temperature.  It  is  always  prefer- 
able, therefore,  to  give  up  the  attempt  to  secure  a  com- 
plete extraction  of  the  starch  and  to  seek  to  control  the 
diastase,  especially  as  the  undissolved  starch  is  not  com- 
pletely lost. 

The  starch  of  malt,  which  escapes  solution  during  sac- 
charification, is  partially  dissolved  during  fermentation. 
High  temperatures  must,  therefore,  be  avoided,  and  sac- 
charification carried  out  between  550  and  6o°. 

In  certain  cases,  and  especially  when  one  is  dealing  with 
raw  materials  of  doubtful  quality  giving  mashes  which  con- 
tain 0.25  to  0.35  per  cent  of  lactic  acid,  it  is  necessary  to  keep 
the  temperature  of  saccharification  still  lower  (not  above 
55°),  because  in  an  acid  medium  the  diastase  becomes  more 
sensitive  to  the  action  of  heat.  In  practice,  unfortunately, 
these  principles  are  entirely  set  aside.  With  a  mouldy  malt 
of  poor  quality  much  higher  temperatures  are  adopted  than 
in  ordinary  work,  because  it  is  supposed  that  by  raising  the 
temperature  the  micro-organisms  which  prevent  the  fermen- 
tation are  killed.  The  results  obtained  by  thus  raising  the 
temperature  are  not  very  satisfactory,  but  the  distiller  is  con- 
soled by  the  thought  that  if  he  had  not  employed  high  tem- 
perature the  final  result  would  have  been  still  worse.     In 


ROLE   OF  AMYLASE  IN    THE  DISTILLERY.  1S7 

reality,  an  increase  of  several  degrees  in  temperature  has  not 
much  influence  on  the  purity  of  the  fermentation  and  does 
not  kill  the  germs  at  all,  but  destroys  the  diastase  and  hinders 
normal  fermentation.  When  working  with  materials  of  poor 
quality,  one  must  have  recourse  to  antiseptics  or  employ  only 
very  active  yeasts  which  can  protect  the  mash  from  the  in- 
vasion of  foreign  ferments  without  hindering  the  secondary 
saccharification. 

The  Infusion  Process. — As  we  have  just  seen,  the  choice 
of  temperature  of  saccharification  presents  great  difficulties. 

The  starch  paste  formed  during  steaming  must  be  lique- 
fied at  a  temperature  higher  than  650. 

The  starch  of  the  malt,  to  be  completely  dissolved,  re- 
quires a  temperature  of  700,  while  the  diastase  cannot  be 
brought  to  a  temperature  of  6o°  without  undergoing  a  per- 
ceptible weakening. 

Under  these  circumstances,  it  is  always  necessary  to  sac- 
rifice either  the  enzyme  or  the  starch,  and  the  temperature  of 
saccharification  must  necessarily  vary  according  to  the  con- 
ditions and  the  quality  of  the  raw  materials. 

An  ideal  process  requires  the  separation  of  the  active  sub- 
stances from  the  starch  of  the  malt  and  their  separate  treat- 
ment at  different  temperatures. 

By  leaving  the  malt  in  contact  with  wat^r  under  suitable 
conditions,  the  diastase  passes  into  solution,  is  separated 
from  the  starch  and  can  serve  afterwards  for  saccharification. 

As  to  the  residue,  it  is  still  impregnated  with  sufficient 
quantities  of  the  enzyme  to  produce  liquefaction.  The 
method,  thus  put  in  practice,  leaves  nothing  to  be  desired. 

The  mashes  cooked  under  pressure  are  liquefied  at  a  tem- 
perature of  700  with  the  soaked  malt  and  then  cooled  to  45°— 
500.  At  that  temperature  the  solution  of  the  enzyme  is 
added;  the  mixture  is  kept  for  some  minutes  at  450— 500 ; 
then  cooled  to  the  temperature  of  fermentation;  the  yeast  is 
added  and  it  is  left  to  ferment. 

This  mode  of  operation  cannot  fail  to  give  good  results 


1 88  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

provided  that  the  extraction  of  the  diastase  has  been  as  com- 
plete as  possible. 

Let  us  now  see  how  one  must  proceed  to  extract  the  dia- 
stase from  the  malt. 

It  is  wrongly  assumed  that  malt  amylase  dissolves  easily 
in  water.  In  reality  the  extraction  is  difficult;  it  depends  on 
the  temperature  of  the  water  and  on  the  thoroughness  with 
which  the  malt  is  ground. 

We  may  emphasize  this  fact  by  the  two  following  experi- 
ments: make  two  mixtures  of  malt  and  water  and  submit 
them  to  a  temperature  of  30°.  Specimen  A  is  not  disturbed, 
while  specimen  B  is  constantly  stirred.  From  time  to  time  a 
few  cubic  centimetres  of  each  liquid  is  taken  and  the  dia- 
static  power  determined,  which  enables  us  to  follow  the 
course  of  the  solution  of  the  diastase. 

Dlastatic  Power. 


Experiments.  After  S  hours,     i- hours.         2c  hours.       4- hours.         52  hours. 

Liquid  A  ^  45  4§  60  55 

B  39  58  52  50  42 

The  quantity  of  diastase  dissolved  in  the  infusion  at  first 
increases  with  the  duration,  reaches  a  maximum,  and  then 
decreases.  In  the  liquid  A  it  is  after  47  hours  that  the  dia- 
static  power  attains  its  maximum.  Shaking  renders  the  ex- 
traction more  rapid  in  the  liquid  B,  where  the  diastatic  power 
reaches  its  maximum  in  17  hours. 

Numerous  experiments  made  with  different  malts  have 
shown  that  this  maximum  is  reached  earlier  as  the  tempera- 
ture of  the  infusion  is  higher.  Our  observations  are  summed 
up  in  the  following  table  : 

An  infusion  prepared  at  450  reaches  its  maximum  of  diastase  dissolved  after  7  or  8  hours. 
li  "  "     from  550  to  59°  "  "  *'  "      3  hours. 

"  "  "        "    6o°  to  65°  "  "  "  "     J£  hour. 

The  time  necessary  for  a  good  extraction  therefore  depends 
on  the  temperature.  There  is.  moreover,  a  critical  time 
which  must  be  borne  in  mind,  since  from  this  time  onward, 
the  diastase  begins  to  disappear. 


ROLE   OF  AMYLASE  IN    THE  DISTILLERY.  189 

The  maximum  quantity  of  active  substance  which  can  be 
dissolved  in  the  infusion  at  the  critical  time  is  not  at  all  con- 
stant. It  varies  considerably  for  the  same  malt  according 
to  the  temperature,  as  may  be  seen  in  the  following  table : 

Diastatic  Power  of  the  Infusion. 


Temperature  of        After  U  hour.        3  hours.         8  hours.        17  hours.        2s  hours. 
Intusion.  '*  J 

30  ..  ..  31  60  49 

45  ..44  56  51 

55  46  55 

65  36  20 

It  is  at  a  temperature  of  300  that  the  most  active  solutions 
are  obtained;  from  450  to  550  the  quantity  of  diastase  which 
can  be  extracted  remains  almost  the  same,  while  at  65 °  the 
destruction  of  the  ferment  occurs  as  it  passes  into  solution, 
and  even  at  the  maximum,  a  very  weak  infusion  is  obtained. 
The  preparation  of  a  cold  infusion  during  17  hours  offers  cer- 
tain practical  difficulties.  To  utilize  the  malt  to  advantage, 
it  is  well  to  make  the  solution  at  55 °  for  3  hours. 

The  infusion  process  is  especially  recommended  in  the 
case  of  malts  of  maize.  These  malts  generally  give  from  8  to 
20  per  cent  of  ungerminated  grains,  and  their  diastatic  power 
is  only  from  one  fifth  to  one  third  that  of  barley-malt.  To 
employ  this  malt  it  is  necessary  to  use  large  quantities,  and 
the  loss  in  starch  is  greater,  because  the  starch  of  maize-malt 
is  much  more  slowly  attacked  by  the  enzyme  than  the  barley- 
starch.  An  infusion  of  this  malt  must  be  made  in  the  follow- 
ing way : 

Reduce  the  malt  to  powder;  dilute  it  in  4-5  volumes  of 
water  at  a  temperature  of  55°.  Then  place  it  in  a  conical  ves- 
sel and  stir  it  during  the  first  hour,  then  leave  it  for  an  hour  or 
an  hour  and  a  half.  Deposition  takes  place  very  readily  and 
the  liquid  can  be  removed  without  carrying  along  the  malt. 

A  filter-press  may  be  used  for  the  same  purpose.  The 
infusion  of  malted  maize  yields  a  liquid  which  filters  easily. 


J 9°  THE  ENZYMES  AND   THEIR  APPLICATIONS. 

The  infusion  of  barley-malt  is  made  in  most  manufactories 
with  crushed  malt,  and  at  a  temperature  of  io°-i5°.  It  is 
prepared  in  a  crushing  apparatus.  The  malt  is  bruised  for 
15-30  minutes,  then  put  in  water  for  one  or  two  hours, 
after  which  the  liquid  which  is  to  be  used  for  saccharification 
is  decanted. 

The  diastatic  power  of  an  infusion  prepared  in  this  way  is 
very  variable.  It  depends  more  upon  the  special  nature  of 
the  malt  than  upon  its  richness  in  amylase.  The  quantity  of 
diastase  extracted  is  between  10  and  50  per  cent  of  that 
contained  in  the  malt.  This  method  of  preparing  an  infu- 
sion of  malt  is  not  one  to  be  recommended. 

Much  more  satisfactory  results  are  obtained  by  prepar- 
ing the  infusion  at  a  temperature  of  45°-50°,  and  allowing 
the  solution  to  proceed  for  2  or  3  hours. 

By  this  method,  from  70  to  80  per  cent  of  the  enzymes 
contained  in  the  malt  are  dissolved.  The  infusion  process 
is  as  yet  scarcely  in  practice,  but  it  is  unquestionably  the 
method  of  the  future. 

The  invention  of  a  contrivance  for  separating  barley-malt 
from  its  infusion  is  to  be  desired,  because  the  principal  dif- 
ficulty always  lies  in  this  operation. 

Concerning  the  Change  which  the  Diastases  Undergo 
during  the  Successive  Stages  of  the  Work. — From  the 
study  we  have  just  made  of  the  conditions  of  the  action  of 
amylase,  it  appears  that  a  part  of  the  enzymes  of  malt  are 
destroyed  during  saccharification  and  that  the  varying  re- 
sistance of  the  diastase  to  temperatures  from  6o°-62°  de- 
pends upon  the  degree  of  acidity  of  the  medium.  The 
acidity  of  the  musts  is  not  the  only  factor  which  produces 
a  change  in  the  diastase ;  other  conditions  must  be  taken  into 
account  and  these  are  not  always  easy  to  recognize. 

Two  malts  possessing  the  same  saccharifying  power, 
used  in  the  same  quantity  and  producing  in  identical  musts 
the  same  quantity  of  sugar  may  nevertheless  yield  infusions 
containing  different  quantities  of  diastase. 


ROLE   OF  AMYLASE  IN    THE  DISTILLERY.  *9l 

Besides  richness  in  active  materials,  other  factors  must 
be  taken  into  consideration  in  estimating  the  value  of  a  malt. 

The  origin  of  differences  in  resistance  is  perhaps  to  be 
found  in  the  degree  of  natural  acidity  of  the  grains,  perhaps 
also  in  the  kind  of  acid  or  in  the  nature  of  other  foreign  sub- 
stances contained  in  the  malt.  We  have  made  a  series  of 
experiments  for  the  purpose  of  finding  the  cause  of  the  dif- 
fering resistance  of  malts  and  we  can  furnish  some  informa- 
tion on  this  subject,  though  unfortunately  it  is  very  incom- 
plete. The  resistance  of  malts  depends  on  the  temperature 
at  which  germination  is  conducted.  Thus,  by  malting  two 
portions  of  the  same  barley  at  different  temperatures,  one 
for  8  days  at  i(f-220,  the  other  for  9  days  at  I2°-I5°,  we 
have  obtained  malts  which  differed  in  their  resistance  at  a 
temperature  of  6o°. 

Malted  barley,  worked  in  the  cold  and  for  9 
hours,  gave  better  results  than  barley  malted  at  higher 
temperatures.  On  the  other  hand  we  found  that  barley, 
giving  from  7  to  10  per  cent  of  non-germinated  grains  after 
malting,  possessed  not  only  a  saccharifying  power  less  than 
that  of  completely  germinated  barley,  but  also  a  widely  dif- 
ferent resistance  to  the  reaction  of  the  medium.  Incom- 
pletely germinated  barley  offers  less  resistance. 

The  richness  of  the  mashes  in  enzymes  after  the  principal 
saccharification  consequently  depends  upon  the  quantity  of 
diastase  which  is  found  in  the  malt,  the  temperature  of 
saccharification,  and  finally  the  degree  of  resistance  of  the 
diastase. 

The  loss  of  enzymes  wnich  occurs  during  saccharification 
at  high  temperatures  may,  under  favorable  conditions,  be 
limited  to  20  per  cent,  but  generally  this  limit  is  exceeded 
and  the  destruction  reaches  30  per  cent. 

The  secondary  saccharification  is  made  with  the  diastase 
which  escapes  destruction  during  the  first  stage  of  the  opera- 
tion.  This  saccharification  is  very  slow  and  must  be  pro- 
longed at  least  three  days. 


19 2  THE  ENZYMES  AND    THEIR   APPLICATIONS. 

The  diastase  is  generally  preserved  much  better  in 
mashes  in  fermentation  than  in  sweet  mashes.  The  dia- 
static  power  of  the  latter  weakens  considerably,  even  in 
the  presence  of  antiseptics.  The  diastatic  power  of  a  mash 
which  has  fermented  under  favorable  conditions  remains 
almost  constant  for  more  than  70  hours.  The  favorable 
progress  of  fermentation  depends  principally  on  the  preser- 
vation of  the  diastase.  This  preservation  can  be  insured 
only  in  musts  free  from  foreign  ferments  and  for  this  reason 
the  use  of  antiseptics  is  necessary  in  the  distillery.  In  fact  it 
is  absolutely  impossible  to  avoid  infection  by  any  other 
means. 

Control  of  the  Process  in  the  Distillery. — The  normal 
progress  of  a  fermentation  depends  upon  various  factors 
and,  besides  those  which  have  to  do  with  the  cooking  and 
the  temperature  of  fermentation  (which  is  always  easy  to 
control),  we  must  take  note  of  the  quality  of  the  malt  used, 
and  of  the  nature  of  the  yeasts  as  well  as  of  the  degree  of 
infection  of  the  must  by  foreign  ferments. 

Each  of  these  three  factors  gives  rise  to  a  problem  which 
is  complicated  by  the  interaction  of  the  others.  And  it  is 
often  very  hard,  when  there  is  some  difficulty  with  the  work,, 
to  recognize  its  cause  and  point  out  its  origin. 

A  poor  fermentation  usually  coincides  with  an  infection 
by  foreign  ferments,  but  this  is  not  always  the  first  cause 
of  the  trouble  observed;  on  the  contrary,  it  is  more  often 
only  the  consequence  either  of  lack  of  diastase  or  of  weak- 
ness of  the  yeast.  Nor  should  the  lack  of  enzymes  in  a  fer- 
menting must  always  be  attributed  to  a  poor  quality  of  malt; 
the  destruction  of  enzymes  may  have  been  caused  by  the 
invasion  of  foreign  ferments.  So,  too,  if  in  a  bad  fermenta- 
tion a  degeneracy  or  a  weakening  of  the  yeast  is  found,  this 
must  not  be  considered  as  the  direct  cause  of  the  trouble; 
the  lack  of  diastase,  among  its  pernicious  effects,  may  have 
brought  the  yeast  into  this  state. 

To  go  back  to  the  beginning  and  discover  the  real  cause 


ROLE   OF  AMYLASE  IN   THE  DISTILLERY.  1 93 

of  the  trouble,  it  is  necessary  to  follow  the  course  of  the 
weakening  of  the  enzyme  in  all  the  stages  of  the  work. 

A  quantitative  determination  of  the  diastase  contained 
in  the  malt  gives  an  idea  of  the  quantity  of  germinated  grain 
necessary  for  a  normal  operation.  Then  by  determining  the 
diastatic  power  of  the  saccharified  mash  produced  with  the 
malt  under  examination,  one  may  find  the  extent  of  the 
change  produced  during  saccharification  and  be  in  a  posi- 
tion to  judge  if  the  quantity  of  amylase  remaining  is  suffi- 
cient for  the  secondary  saccharification.  The  determination 
of  the  diastatic  power  of  the  mash  at  different  stages  of  fer- 
mentation furnishes  data  on  the  weakening  of  the  diastase; 
it  makes  it  possible  to  ascertain  the  point  at  which  activity 
begins  to  diminish  and  to  recognize  clearly  the  cause  of 
this  diminution.  A  perceptible  weakening  of  the  enzyme  in 
the  first  period  of  fermentation  must  lead  to  an  irregular 
progress.  The  cause  of  the  phenomenon  is  generally  the 
initial  acidity  of  the  must  and  it  is  well,  in  such  cases,  to 
choose  a  saccharification  temperature  much  below  6o°. 

The  weakening  of  the  diastase  during  fermentation  may 
be  due  to  other  causes.  It  may  come  from  the  quality  of 
the  grain  and  in  this  case  high  pressure  during  cooking 
must  be  avoided,  because  it  is  generally  during  this  process 
that  substances  which  weaken  the  diastase  are  formed. 

During  the  secondary  fermentation,  the  acidity  must  be 
determined  at  the  same  time  with  the  weakening  of  the  dia- 
stase, for  a  perceptible  increase  in  acidity  is  always  followed 
by  partial  destruction  of  the  diastase.  The  weakening  of 
the  diastase  may  be  averted  in  this  case  by  an  addition  of 
antiseptics. 

An  entirely  opposite  state  of  affairs  is  sometimes  ob- 
served: the  diastase  first  weakens  and  acidification  is  pro- 
duced only  from  6  to  10  hours  later. 

The  appearance  in  the  musts  of  foreign  ferments  here 
results  from  weakening  of  the  diastase.     In  this  case  the 


194  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

addition  of  more  of  the  infusion  to  the  fermenting  must 
may  prevent  acidity  and  keep  up  the  yield  of  alcohol. 

Finally,  if  an  incomplete  fermentation  is  met  with  in  the 
musts  which  are  not  abnormally  acid  and  are  rich  in  diastase, 
the  cause  may  lie  in  the  yeasts. 

This  case  often  arises  when  antiseptics  are  used  which 
leave  the  diastase  intact,  but  act  very  unfavorably  on  certain 
kinds  of  yeasts. 

From  the  foregoing,  it  is  evident  that  determinations  of 
the  fermenting  power  of  malt  and  of  musts  may  be  of  great 
service  to  distillers.  In  the  following  chapter  will  be  found 
the  methods  to  be  followed  in  such  analyses. 

BIBLIOGRAPHY. 

Effront. — Sur  les  conditions  chimiques  de  Taction  des  diastases.  Comptes- 
Rendus,  1892,  t.  115,  p.  1524. 

■ Sur  certaines  conditions  chimiques  de  Taction  des  Ievures  de  biere. 

Comptes   Rendus,   1893,  t.   117,  p.   559. 

Sur  la  formation  de  Tacide  succinique  et  de  la  glycerine  dans  la  fer- 
mentation alcoholique.     Comptes  Rendus,   1894,  t.   119,  p.  92. 

Accoutumance  des  ferments  aux  antiseptiques  et  influence  de  cette 

accoutumance  sur  leur  travail  chimique.  Comptes  Rendus,  1894,  t. 
119,  p.  169. 

De    Tinfluence    des    composes    du    fluor    sur    les    Ievures    de    biere. 

Comptes  Rendus,  1894,  t.   118,  p.  1420. 

Etude  sur  les  Ievures  lactiques.     Annales   de  l'Inst.     Pasteur,   1896, 

p.   S24. 

De  Tinfluence  des  fluorures  sur  Taccroissement  et  le  developpement 

des  cellules  de  la  levure  alcoholique.  Moniteur  scientifique,  1891, 
P;   254. 

Etude  sur  les  Ievures.     Monit.  scientifique,  XI,  p.  1138,  1891. 

Des   conditions   auxquelles   doivent    satisfaire  les   solutions   fermen- 

tescibles  pour  que  les  fluorures  y  produisent  un  maximum  d'effet. 
Monit.  scientifique,  1892,  t.  VI,  p.  81. 

Maercker. — Spiritusfabrikation.     Paul  Parey,  Berlin,  1894. 

Max  Biicheler.  Die  Branntwein  Industrie.  Zweite  vollstandig  umgear- 
beitete  Auflage  des  Lehrbuches  der  Branntweinbrennerei  von  Stam- 
mer.    Braunschweig. 

Leitfaden  fur  den  landwirthschaftlichen  Brennereitreib.  Braun- 
schweig, 1898. 


CHAPTER  XVI. 

QUANTITATIVE  STUDY  OF  MALT. 

Determination  of  the  diastatic  power  of  malt  and  mashes  by  the  methods 
of  Effront. — Determination  of  saccharifying  and  liquefying  powers. — 
Determination  of  the  diastatic  power  of  sweet  and  fermented  mashes. 

The  methods  generally  used  for  the  quantitative  study  of 
malt  take  account  only  of  its  saccharifying  power  and  en- 
tirely neglect  its  liquefying  power  as  well  as  the  resistance  of 
the  enzymes.  Our  researches  have  shown  that  it  is  indis- 
pensable to  take  account  of  these  two  properties.  The  sac- 
charifying power  of  malt  is  subject  to  the  influence  of  foreign 
substances  contained  in  the  grains.  The  intensity  of  the  sac- 
charifying power  of  a  malt  does  not,  therefore,  afford  an 
exact  measure  of  the  quantity  of  amylase  it  contains.  The 
effect  obtained  in  a  saccharirication  by  diastase  is  often  the 
result  of  the  combined  action  of  the  enzyme  and  of  accelerat- 
ing substances  which  accompany  it. 

The  following  experiment  gives  a  clear  idea  of  the  influ- 
ence of  these  extractive  materials  of  the  grain  on  the  saccha- 
rifying power: 

Prepare  an  infusion,  using  one  part  of  malt  and  twelve 
parts  of  water,  and  at  the  same  time  an  infusion  of  non- 
malted  barley  with  one  part  of  the  grain  and  four  parts  of 
water.  Filter  these  two  infusions:  from  each  take  a  certain 
number  of  cubic  centimetres,  which  are  introduced  into  a 
starch  paste.  Saccharify  for  one  hour  at  500.  The  quantity  of 
maltose  obtained  under  these  conditions  furnishes  a  basis  of 
comparison  between  the  diastatic  value  of  the  two  infusions. 

195 


196 


THE  ENZYMES  AND    THEIR.  APPLICATIONS. 


In  a  second  series  of  experiments,  add  to  the  starch,  at 
the  same  time  with  the  infusions  of  malt  and  of  fresh  barley, 
a  certain  quantity  of  infusion  previously  boiled. 

The  saccharification  is  made  at  the  same  temperature  in 
all  the  experiments. 


Number  of  the 
Experiment. 

Fresh 
Infusion. 

1  C.C. 

2  " 
6      " 

0  " 

1  " 
I      " 

Boiled 
Infusion, 

Maltose 
formed. 

' 

I 
2 
3 
4 
5 
6 

0.37    g- 
O.65     " 

O.85     " 
0           " 
O.6       " 
O.72     " 

I 

6  c.c. 
r    " 
2    " 

r 
1 

*7 
/ 

8 

9 
10 

0.5  C.C. 

0.5  " 

0      " 

0.5  " 

2   c.c. 

o-5  " 
1     " 

0.07    g. 
O           " 

1 
1 

O.O95" 
O.  II    " 

Six  cubic  centimetres  of  the  barley  infusion,  not  boiled, 
give  in  the  starch  0.85  g.  of  maltose  (Experiment  No.  3), 
The  same  quantity  of  infusion,  previously  boiled,  used  with- 
out fresh  infusion,  remains  absolutely  without  action  on  the 
starch  (Experiment  No.  4).  But  this  liquid,  inactive  by  it- 
self, influences  saccharification  to  a  great  degree  if  it  is  with 
active  diastase.  Thus,  a  cubic  centimetre  of  infusion  of  bar- 
ley gives  0.37  g.  of  maltose  and  this  same  quantity  of  in- 
fusion produces  0.72  g.  of  maltose  when  it  has  2  cubic  cen- 
timetres of  boiled  infusion  added. 

The  same  thing  is  true  with  an  infusion  of  malt  heated  to 
ioo°;  0.5  cubic  centimetres  of  this  infusion  furnish  0.07  g. 
of  maltose  and  the  same  quantity  of  infusion  gives  o.  11 
g.  of  maltose  when  saccharification  occurs  with  a  cubic  cen- 
timetre of  boiled  infusion. 

Thus  it  is  seen  that  the  extractive  materials  of  raw  grain 
have  a  considerable  action  on  the  amylase  of  malt  and  that 
with  them  saccharification  can  produce  ten  times  as  much 
sugar  as  by  the  action  of  the  ferment  alone. 

Analogous  experiments  with  barley  of  different  origin 


QUANTITATIVE  STUDY   OF  MALT.  197 

have  shown  us  that  no  constant  ratio  exists  between  the  real 
saccharifying  power  (due  to  amylase)  and  the  accelerating 
power  latent  in  raw  grain.  It  follows  that  the  active  constit- 
uents of  grains  of  different  origin  influence  the  saccharifying 
power  to  varying  degrees. 

It  must  be  admitted  that  in  ascertaining  the  value  of  a 
malt  it  makes  no  difference  whether  the  saccharifying  power 
comes  from  the  amylase  or  some  other  substance,  but  amy- 
lase must  not  be  confounded  with  the  substances  which  ac- 
celerate saccharification;  the  mode  of  action  of  the  latter  is 
wholly  different  from  that  of  the  diastase.  The  substances 
which  excite  or  accelerate  hydration  do  not  always  increase 
the  quantity  of  sugar  formed  and  they  have  absolutely  no  in- 
fluence on  the  distillery  mash.  In  practice, — and  that  is  the 
important  point  for  us, — it  is  amylase  alone  which  comes 
into  play. 

This  results  from  the  fact  that  the  exciting  substances  act 
only  in  mashes  containing  little  maltose,  and  because  the  ef- 
fect they  produce  becomes  weaker  and  weaker  as  sacchari- 
flcation advances.  In  distillery  and  brewery  mashes  a  large 
proportion  of  maltose  is  always  present  and  the  effect  of 
exciting  substances  is  negligible. 

The  determination  of  the  amylase  of  malt,  looking  solely 
to  its  saccharifying  power,  will  consequently  always  give  un- 
reliable results.  To  reach  more  trustworthy  results,  we  have 
sought  a  method  which  will  allow  of  measuring  quantitatively 
the  liquefying  power.  The  liquefying  power  of  malt  is  not 
influenced  by  foreign  substances.  For  this  reason  we  ex- 
press the  value  of  a  malt  by  both  its  powers :  its  saccharify- 
ing power  and  its  liquefying  power. 

In  the  preceding  chapter  we  have  seen  that  malts  differ 
much  in  their  resistance  to  a  temperature  of  6o°.  This  cir- 
cumstance forces  us,  in  determinations,  to  take  account  of 
the  degree  of  resistance  of  the  amylase.  These  factors  be- 
come especially  important  in  the  case  of  malts  destined  for 
the  distillery. 


I98  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

A  malt  of  high  diastatic  power,  but  of  little  resistance  to 
high  temperatures,  gives  a  less  satisfactory  result  in  the  dis- 
tillery than  a  malt  less  rich  in  amylase  but  which  endures 
without  change  a  high  temperature  of  saccharification. 

To  form  an  idea  of  the  greater  or  less  resistance  of  malt 
at  a  high  temperature,  we  keep  the  malt  for  an  hour  at  6o° 
and  determine  the  saccharifying  power  in  mashes  where  the 
diastase  has  previously  been  destroyed. 

We  have  thus  established  a  method  of  determination 
which,  we  believe,  answers  to  the  needs  of  the  industry.  The 
determination  is  made  in  three  stages : 

1st.   Preparation  of  an  infusion. 

2nd.   Determination  o'f  the  saccharifying  power. 

3rd.   Determination  of  the  liquefying  power. 

Preparation  of  the  Infusion. — To  prepare  the  infusion, 
weigh  out  6  grams  of  crushed  malt,  put  them  in  a  flask  con- 
taining 100  cubic  centimetres  of  water  at  6o° ;  keep  the  flask 
in  a  water-bath  for  an  hour  at  a  temperature  of  6o°.  During 
saccharification,  shake  the  flask  from  time  to  time ;  the  sac- 
charincation ended,  cool  to  300  and  filter;  to  50  cubic  cen- 
timetres of  filtered  liquid  then  add  50  cubic  centimetres  of 
distilled  water  and  determine  the  saccharifying  power  of  this 
diluted  infusion.  The  remainder  of  the  non-diluted  infusion 
is  used  to  determine  the  liquefying  power. 

Determination  of  the  Saccharifying  Power. — The  sac- 
charifying power  is  determined  by  the  aid  of  a  standard  solu- 
tion of  starch. 

Dissolve  2  grams  of  starch  in  water  and  make  the  solu- 
tion up  to  100  cubic  centimetres.  To  100  cubic  centimetres 
of  this  2  per  cent  solution,  add  55  cubic  centimetres  of 
distilled  water  and  5  cubic  centimetres  of  an  infusion  of  malt 
diluted  as  described  above.  The  whole  is  then  placed  in  a 
water-bath  at  6o°  for  an  hour. 

After  saccharification,  it  is  rapidly  cooled  and  the  content 
of  sugar  immediately  determined. 

To  determine  the  maltose  in  the  saccharine  liquid,  2  cubic 


QUANTITATIVE  STUDY   OF  MALT.  ^99 

centimetres  of  a  solution  of  cupro-potassic  tartrate  is  used, 
which  corresponds  to  0.01498  grams  of  maltose.  The  2 
cubic  centimetres  of  cupro-potassic  solution  is  put  in  a  test- 
tube  to  which  is  added  3  cubic  centimetres  of  water  and  a  few 
fragments  of  pumice-stone.  The  number  of  cubic  cen- 
timetres of  saccharine  solution  necessary  for  the  reduction  of 
the  copper  salt  varies,  according  to  the  malts,  from  3  to  20; 
comparative  experiments  have  shown  that  where  3  to  5  cubic 
centimetres  of  the  saccharified  solution  reduce  2  cubic  cen- 
timetres of  cupro-potassic  tartrate  under  the  conditions  in- 
dicated, the  malt  may  be  considered  as  having  a  maximum 
saccharifying  power ;  6  to  8  cubic  centimetres  correspond  to 
a  good  malt,  9  to  12  to  a  malt  of  medium  value,  and  if  the 
quantity  of  saccharified  solution  necessary  for  the  reduction 
is  from  14  to  20  cubic  centimetres,  the  malt  may  be  consid- 
ered poor. 

The  small  quantity  of  maltose  introduced  with  the  in- 
fusion does  not  have  much  influence  on  the  results,  which, 
moreover,  do  not  serve  as  a  basis  for  accurate  calculations, 
but  merely  as  supplementary  data  for  the  estimation. 

The  standard  starch  solution  which  we  use  for  the  deter- 
mination of  the  saccharifying  power  is  prepared  in  the  follow- 
ing manner :  Let  potato-flour  soak  at  a  temperature  of  400 
in  a  solution  of  7  per  cent  hydrochloric  acid,  shaking  the 
liquid  every  6  hours. 

After  3  days  decant  the  liquid,  wash  the  mass  with  water 
to  a  neutral  reaction,  and  dry  at  the  ordinary  temperature. 

The  product  obtained  contains  from  \y\  to  18  per  cent  of 
water  and  is  completely  dissolved  in  warm  water. 

By  submitting  to  this  operation  different  potato-flours  of 
the  same  origin,  the  same  product  is  constantly  obtained,  but 
the  result  differs  much  when  starches  from  different  sources 
are  used,  as  the  diastase,  in  this  case,  shows  itself  more  active 
in  some  samples  than  in  others. 

It  is  necessary  that  the  standard  starch,  before  being  used 
for  a  determination,  should  be  tried  with  a  malt  of  known 


200  THE  ENZYMES  AND    THEIR   APPLICATIONS. 

saccharifying  power.  If  the  same  power  is  found  in  the 
starch  thus  tested,  this  can  be  considered  as  a  standard  mix- 
ture. Otherwise,  one  must,  by  repeated  trials,  determine 
the  composition  of  a  new  starch  solution  which  must  be 
taken  to  replace  2  grams  of  the  standard. 

Given,  for  example,  a  malt  which  possesses  a  saccharify- 
ing power  of  4.5,  measured  by  the  standard  starch,  we  will 
suppose  that  the  same  malt  with  another  starch  mixture 
would  have  a  saccharifying  power  of  4.1.  The  question  is  to 
determine  what  quantity  of  the  new  starch  must  be  taken  to 
give  the  same  result  as  2  grams  of  the  standard.  To  this  end 
solutions  are  prepared  which  contain,  instead  of  2  grams  per 
100  of  starch,  1.9,  1.8,  1.7  grams  per  100,  and  the  sacchari- 
fying power  of  the  malt  is  tried  with  these  solutions.  If  it  is 
found  that  in  the  solution  containing  1.7  g.  of  starch  the 
saccharifying  power  is  4.5,  1.7  g.  of  the  new  starch  will  be 
constantly  used  instead  of  2  grams,  and  under  these  condi- 
tions alone  can  it  be  used  in  place  of  the  standard.  One  must 
always  use  a  fresh  starch  solution,  for  we  have  found  that  this 
solution,  although  it  keeps  fairly  well,  acts  differently  with 
the  same  malt,  according  as  it  is  fresh,  or  has  been  prepared 
for  some  time. 

This  peculiarity  is  the  more  unexpected  since  we  find  no 
difference  in  acidity  in  the  two  solutions  of  starch. 

Determination  of  the  Liquefying  Power. — Weigh  out  40 
grams  of  standard  rice-starch;  dilute  with  a  little  water  in  a 
capsule;  introduce  the  mixture  into  a  100  cubic  centimetre 
calibrated  flask ;  rinse  the  capsule  with  a  fresh  portion  of 
water  which  is  poured  into  the  flask  and  make  the  volume  up 
to  100  cubic  centimetres.  From  the  mixture  of  starch  and 
water  briskly  stirred,  take  with  a  pipette  8  specimens  of  5 
cubic  centimetres  each,  and  introduce  them  into  numbered 
test-tubes  of  10  cubic  centimetres  capacity;  add  to  the  con- 
tents of  each  tube  the  same  quantity  of  infusion  of  malt  pre- 
pared in  the  manner  indicated  above.  For  each  of  the  num- 
bered tubes,  containing  2  grams  of  starch  and  the  infusion, 


QUANTITATIVE  STUDY  OF  MALT.  201 

prepare  a  second  larger  tube  having  a  diameter  of  19  milli- 
metres, a  height  of  19  centimetres  and  similarly  numbered. 
In  each  of  the  large  tubes  place  14  cubic  centimetres  of  dis- 
tilled water,  and  put  them  in  a  water-bath  at  8o° ;  then  bring 
them  one  after  another  rapidly  to  the  boiling  point,  and  pour 
into  the  boiling  liquid  the  starch  paste  and  infusion  contained 
in  the  smaller  tube  having  the  same  number.  Stir  rapidly 
with  a  glass  rod,  rinse  the  tube  which  contained  the  starch 
with  a  cubic  centimetre  of  water  and  add  it  to  the  contents 
of  the  large  tube.  Stir  again  with  the  rod,  mark  the 
hour  exactly  and  leave  in  the  water-bath  at  8o°  for  10 
minutes.  Take  them  one  by  one,  stir  the  contents  once  more 
with  a  glass  rod  and  plunge  them  into  a  water-bath  at  ioo°, 
where  they  stay  exactly  10  minutes.  After  this  operation  all 
the  tubes  are  rapidly  cooled.  A  thermometer  placed  in  one 
of  them  indicates  the  moment  when  the  temperature  reaches 
1 5°,  and  it  is  at  this  point  that  the  degree  of  liquefaction  is 
ascertained.  The  tubes,  thus  cooled  to  150,  are  inverted  one 
after  the  other.  If  the  contents  of  the  tube  runs  out  in- 
stantly and  without  difficulty,  the  specimen  is  considered 
liquefied;  a  tube  which  empties  entirely,  but  whose  contents 
presents  the  consistency  of  a  thick  syrup,  shows  a  three-quar- 
ters liquefaction ;  a  tube  which  does  not  become  entirely 
empty  shows  a  partial  liquefaction. 

If  the  tube  whose  contents  are  entirely  liquid  has,  for  ex- 
ample, received  2  cubic  centimetres  of  the  non-diluted  in- 
fusion, the  liquefying  power  is  expressed  by  2. 

Comparative  experiments  with  different  malts  have  shown 
that  a  liquefying  power  of  1.5  to  2  shows  a  malt  of  excellent 
quality.  A  liquefying  power  of  2.5  to  3  corresponds  to  a 
malt  of  good  quality,  while  a  liquefying  power  of  3.5  to  4 
shows  a  malt  of  doubtful  quality  whose  value  will  depend  011 
its  saccharifying  power.  A  malt  with  a  liquefying  power  of 
4  and  a  saccharifying  power  of  4  to  5  does  passable  work  in 
the  distillery,  while  a  malt  having  the  same  liquefying  power 
as  the  preceding  and  a  saccharifying  power  of  7  to  9  must  be 


202  THE  ENZYMES  AND    THEIR   APPLICATIONS. 

considered  poor.  The  difference  between  the  saccharifying 
and  liquefying  powers  is  especially  marked  in  dry  malt.  By 
drying,  the  saccharifying  power  is  considerably  weakened, 
while  the  liquefying  power  is  much  less  changed.  In  the  dry 
malt  a  liquefying  power  of  2  to  3  does  not  necessarily  prove 
an  excellent  product :  all  depends  on  the  saccharifying 
power.  With  a  moderate  saccharifying  power  the  malt  may 
hiave  a  great  value,  but  with  a  saccharifying  power  of  12  the 
malt,  even  when  it  has  a  great  liquefying  power,  cannot  be 
used  in  the  distillery. 

To  determine  the  liquefying  power,  rice-starch,  chosen 
with  great  care,  is  used.  The  rice-starches  of  different 
origin  act  differently  with  diastase  at  8o°  ;  so  rice-starches 
xnay  be  grouped  in  two  classes.  In  the  first  are  placed  the 
products  which,  at  the  moment  of  liquefaction,  become  com- 
pletely colorless  and  transparent ;  in  the  second,  those  which 
preserve  a  whitish  tint  and  give  an  opaque  liquefaction.  The 
ffrst  liquefy  with  much  more  difficulty  than  the  second  and 
the  liquefying  power  of  an  infusion  may  vary  much  according 
as  one  or  the  other  type  of  starch  is  used. 

At  the  beginning  of  our  investigation  we  worked  with  a 
starch  of  the  second  type;  we  afterwards  abandoned  it  be- 
cause we  found  that  the  starches  which  give  a  transparent 
liquefaction  are  preferable,  the  exact  time  of  liquefaction 
being  more  easy  to  observe. 

If  one  wishes  to  obtain  constant  results  the  same  starch 
must  always  be  used  in  testing  amylase. 

As  a  standard  we  use  Hoffman  starch  and  whenever  we 
make  a  new  mixture  we  verify  the  starch  with  the  standard 
specimen.  The  verification  is  made  with  infusion  of  malt. 
Two  grams  of  standard  starch  and  2  grams  of  the  starch  to 
be  tested  are  liquefied  with  the  same  quantity  of  infusion  at  a 
temperature  of  8o°  for  ten  minutes.  If  the  number  of  cubic 
centimetres  of  infusion  necessary  to  liquefy  completely  the 
standard  starch  and  the  starch  to  be  tested  is  the  same,  the 
two  starches  may  be  considered  identical.     Otherwise,  in- 


QUANTITATIVE  STUDY  OF  MALT.  203 

crease  or  diminish  the  quantity  of  starch  tested  in  such  a  way 
as  to  obtain  liquefaction  with  the  same  quantity  of  infusion. 

If  it  happens,  for  example,  that  for  the  liquefaction  of 
2  grams  of  standard  starch  it  takes  2.5  c.c.  of  an  infusion  of 
malt  and  that  for  the  liquefaction  of  the  same  quantity  of 
starch  under  examination  it  takes  3  cubic  centimetres  of  the 
same  infusion,  one  must  weigh  out  1.9  gr.,  1.8  gr.,  1.7  gr.,  of 
the  starch  to  be  tested  and  see  which  of  these  amounts  is 
liquefied  by  2.5  c.c.  of  infusion.  If  complete  liquefaction  is 
produced  by  1.9  gr.,  it  must  be  concluded  that  instead  of  2 
grams  of  standard  starch  one  must  take  only  1.9  gr.  of  the 
starch  which  is  being  tested. 

Another  method  for  transforming  a  certain  starch  into 
standard  starch  consists  in  acidifying  it  or  making  it  alkaline. 

This  method,  which  is  preferable  to  the  other,  has  for  its 
basis  the  following  observation  : 

The  standard  starch  is  slightly  alkaline,  and  if  rice-starch 
is  brought  to  the  same  degree  of  alkalinity  it  acquires  all  the 
properties  of  the  standard  starch. 

The  liquefying  power  is  so  sensitive  to  the  alkalinity  of 
the  liquid  that  the  quantity  of  soda  to  be  added  cannot  be 
determined  by  a  single  alkalimetric  measurement.  If  the 
difference  in  alkalinity  of  the  two  starches  corresponds  to 
2  cubic  centimetres  of  decinormal  solution  of  soda,  one  must 
add  only  half  of  the  alkaline  solution  for  50  grams  of  starch, 
and  then  keep  adding  tenths  of  cubic  centimetres  until  the 
two  starch  pastes  liquefy  with  the  same  quantity  of  infusion 
of  malt. 

The  standard  starch  for  saccharification,  as  well  as  the 
standard  for  liquefaction  *  keeps  without  change  in  bottles 
with  ground  stoppers  and  can  be  used  for  determinations, 
for  at  least  two  years. 

We  have  also  observed  that  dry  malt,  kept  in  the  same 


*  Standard  starches  which  we  use  are  for  sale  at  Drosten's,  rue  du 
Marais,  Brussels,  and  at  H.  Koenig's,  manufacturer  of  chemicals,  Leipzig. 


204 


THE  ENZYMES  AND    THEIR  APPLICATIONS. 


way,   preserves   for  years  its  saccharifying   and   liquefying- 
powers. 

We  give  here  two  determinations  of  malt  made  accord- 
ing to  the  method  indicated  above. 

Malt  A. — Russian  barley,  soaked  2\  days  with  aeration,, 
malted  in  revolving  drums. 

Minimum  temperature  i8°,  maximum  21  °. 
Duration  of  germination,  4  days. 
Water  48.04%. 
General  aspect  and  odor,  normal. 

3  non-germinated  grains. 
34  grains  whose  plumule  was  shorter  than- 
the  grain, 
having  a  plumule  of  the  length  of 

the  grain, 
having  a  plumule  i^  times  as  long 

as  the  grain, 
having  a  plumule  more  than  twice 
as  long  as  the  grain. 
The  liquefying  power  and  the   saccharifying  power  of 
this  malt  were  determined  in  three  different  specimens: 
1st.   In  unsorted  grains. 
2nd.  In  grains  in  which  the  plumules  are  twice  as  long  as 
the  grains. 

3rd.  In  grains  in  which  the  length  of  the  plumules  does- 
not  exceed  that  of  the  grain. 


100  grains 
include 


30 


21 


12 


Unsorted  Malt. 

Malt  with  Plumule 

twice  the  Length 

of  the  Grains. 

Malt  with  Plumule^ 

not  longer  than 

Grains. 

0.6 

O.585. 

0.53 

17.  C.C. 

9.5  c.c. 

20.7  C.C. 

f 

L 

2.5  c.c.  not  liq. 

3  "       "     " 
3-5    "     .  i." 

4  "    liquid 

2.5  c.c.  not  liq. 
3        "       "      " 
3.5    "     liquid 

3.5  c.c.  not  liq- 
4       "      i     " 

QUANTITATIVE  STUDY  OF  MALT.  205 

The  maltose  indicated  for  each  infusion  shows  the  sugar 
content  of  the  dilute  infusion  which  was  used  for  determining 
the  saccharifying  power. 

In  the  grains  not  classified,  when  coming  out  of  the 
apparatus,  there  is  found  a  saccharifying  power  of  17  and  a 
liquefying  power  of  4.  Therefore  the  malt  is  decidedly  me- 
diocre; the  trial  of  fermentation  with  different  quantities 
of  malt  has  shown  us  that  it  takes  18  parts  of  this  malt  per 
100  of  rice  to  insure  a  complete  transformation. 

The  comparative  analysis  of  the  three  specimens  confirms 
for  us  the  facts  established  by  the  Institute  of  Berlin,  namely, 
that  the  development  of  the  plumules  coincides  with  an  in- 
crease in  the  quantity  of  the  enzymes. 

Malt  B. — Saladin  pneumatic  system  malt-house.  Malt- 
house  of  Buir,  near  Cologne. 

Small  Russian  barley,  soaked  2\  days  without  aeration. 

Duration  of  malting,  9  days. 

Temperature,  minimum,  18°;  maximum,  230. 

The  sprouting  is  uniform  and  the  plumules  do  not  ex- 
ceed the  grain. 

Non-germinated  grains,  3. 

Water,  47%. 

Maltose  of  infusion,  0.74. 

Saccharifying  power,  4.65. 

25  c.c.  =  liquid. 


Liquefying  power  . 

(2  c.c.  =  /a  liquid. 

The  liquefying  power  as  well  as  the  saccharifying  power 
shows  a  malt  of  excellent  quality.  The  quantity  of  malt 
necessary  for  fermenting  100  kilograms  of  rice  is  8  kilo- 
grams. 

The  method  we  have  just  indicated  is  applicable  to  the 
investigation  of  malts  of  barley  and  rye. 

For  the  analysis  of  malt  of  maize  other  conditions  are 
necessary  because  this  malt  always  contains  relatively  small 
quantities  of  diastase.  For  the  preparation  of  the  infusion 
12  grams  of  ground  malt  are  taken  instead  of  6,  and,  to  de- 


206  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

termine  the  saccharifying  power,  only  i  cubic  centimetre  in- 
stead of  2  of  the  cupro-potassic  solution  is  used. 

A  maize  malt  of  fine  quality  always  contains  4  to  8  per 
cent  of  non-germinated  grains.  It  possesses,  under  the  con- 
ditions indicated,  a  saccharifying  power  of  4  to  6  and  a  lique- 
fying power  of  2.5  to  3. 

By  comparing  the  malt  of  maize  with  barley  malt  of 
first  quality,  it  is  found  that  it  is  only  one  fourth  as  active  and 
in  practice  it  is  necessary  to  use  4  to  5  times  as  much  of  this 
as  of  barley-malt  to  secure  the  same  result. 

Method  of  Analysis  of  Sweet  and  Fermented  Mashes. 
— The  saccharifying  power  of  the  mash  may  be  determined 
by  aid  of  the  coloration  which  the  mash  gives  with  iodine. 
This  is  done'  in  the  following  manner:  take  6  specimens  of 
20  cubic  centimetres  each  of  a  fresh  2%  solution  of  soluble 
starch  and  place  each  sample  in  a  numbered  test-tube;  add 
with  a  pipette  divided  into  tenths  of  a  cubic  centimetre 
.25  c.c,  .50  c.c,  .75  c.c,  1  c.c,  1.25  c.c,  and  1.5  c.c,  of  the  sac- 
charine or  fermented  must  to  be  tested;  place  the  tubes  in  a 
water-bath  at  6o°  for  an  hour;  cool  and  add  to  the  contents 
of  each  tube  half  a  cubic  centimetre  of  a  very  dilute  solution, 
of  iodine  and  observe  the  coloration  at  the  moment  the 
iodine  is  added  to  the  liquid. 

A  saccharification  made  with  .25  c.c.  of  sweet  mash,  not 
colored  by  iodine,  corresponds  to  the  maximum  of  sacchari- 
fying power,  and  if  this  result  is  reached,  one  may  be  sure 
that  a  greater  quantity  of  malt  than  necessary  has  been  used 
for  the  fermentation.  The  absence  of  coloration  in  a  tube 
which  has  received  .75  c.c.  of  solution  shows  that  the  sweet 
mash  possesses  a  saccharifying  power  sufficient  for  fermen- 
tation, at  least  if  the  liquefying  power  of  this  must  is  normal. 

If  a  coloration  appears- in  the  liquid  containing  1.25  c.c. 
of  the  saccharified  mash,  it  is  certain  that  this  mash  does  not 
contain  the  necessary  quantity  of  malt  and  it  is  useless  to 
pay  any  attention  to  the  liquefying  power.  Failure  to  give 
color  with  iodine  of  solutions  which  have  received  1  cubic 


QUANTITATIVE  STUDY  OF  MALT.  207 

-centimetre  of  mash  shows  a  sufficient  quantity  of  diastase  if 
the  liquefying  power  is  very  great.  Otherwise,  the  saccha- 
rified mash  is  not  rich  enough  in  active  materials. 

This  method  is  of  great  service  for  the  control  of  mashes 
during  fermentation;  the  saccharifying  power  is  determined 
by  coloration  with  the  solution  of  iodine  at  the  beginning 
of  fermentation,  and  this  operation  is  repeated  after  30  and 
60  hours.  The  saccharifying  power  determined  by  this 
method  at  the  beginning  of  fermentation  should  not  change 
much  up  to  the  end  of  the  operation.  If  it  is  found  that,  at 
a  certain  time,  twice  as  much  liquid  is  needed  as  at  the  be- 
inning  to  have  no  coloration  with  iodine,  one  may  be  cer- 
tain that  there  is  a  change  in  the  diastase  and  it  is  important 
to  add  more  infusion. 

A  mash  fermented  for  86  hours  should  have  a  sacchari- 
fying power  of  between  0.75  and  1,  that  is,  with  0.75  to  1 
cubic  centimetre  of  mash  no  coloration  by  iodine  should  be 
obtained.  A  saccharifying  power  of  1.5  at  the  end  of  fer- 
mentation shows  a  lack  of  diastase. 

These  data  are  applicable  to  mashes  of  rice  of  a  density 
of  17  to  19  Balling.  Mashes  of  grains  and  potatoes  act  dif- 
ferently. In  these,  the  weakening  of  the  diastases  during 
saccharification  and  the  fermentation  occur  much  more  rap- 
idly. One  must  seek  to  have  mashes  of  a  saccharifying 
power  of  I  to  1.25  at  the  beginning  of  the  operation  and 
of  2  at  the  end  of  fermentation. 

Our  method  of  determination  of  malt  and  mash  is  now 
introduced  in  the  central  stations  of  the  Association  of  Dis- 
tillers of  Bavaria  and  Austria-Hungary.  The  directors  of 
these  stations,  Professors  Kruis  and  Biicheler  'have  ex- 
pressed to  us  their  entire  satisfaction. 

With  a  little  experience,  one  can  succeed  in  having  a 
complete  control  of  the  work  by  means  of  this  method. 

BIBLIOGRAPHY. 

J.    Effront. — Contributions   a   l'etude   de    l'amylase.      Monit.    scientifique, 
tome  VIII,  p.  541,  et  tome  X,  p.  711. 


CHAPTER  XVII. 

MALTASE. 

Glucase  of  Cusenier. — Maltase  of  yeast. — Properties. — Differences  be- 
tween the  optimum  temperatures  of  different  glucases. — Maltase  of 
moulds. — Manner  of  action  upon  starch. — Processes  of  secretion. — In- 
fluence of  nitrogenous  food. — Influence  of  carbohydrates. — The  dif- 
ferent amylomaltases  of  Laborde. 

Maltase  or  glucase  is  an  enzyme  which  acts  upon  starch, 
dextrins,  and  maltose. 

The  existence  of  an  enzyme  acting-  on  maltose  was 
doubted  for  a  long  time.  It  is,  however,  evident  that  mal- 
tose, to  be  assimilated  by  living  cells,  must  be  hydrated  and 
transformed  into  glucose. 

In  1865,  Bechamp  found  in  the  urine  the  presence  of  an 
enzyme  acting  on  maltose  which  he  called  nefrozymase. 
Brown  and  Heron  discovered  an  analogous  active  principle 
in  the  pancreatic  juice  and  the  small  intestine  of  the  pig. 
Later  Emile  Bourquelot  confirmed  the  observation  of  Brown 
and  Heron,  demonstrating  the  presence  of  the  same  principle 
in  the  pancreas  and  small  intestine  of  the  rabbit. 

The  diastatic  liquids  obtained  by  these  workers  presented 
very  varied  properties.  They  evidently  contained  diastases 
of  very  different  natures  and  it  was  very  difficult  to  establish 
definitely  the  presence,  in  the  liquids  studied,  of  a  special 
ferment  acting  solely  upon  maltose. 

The  discovery  of  the  active  principle  which  decomposes 
maltose  into  two  molecules  of  glucose  dates  from  1886.  It 
belongs  to  Leon  Cusenier,  who  named  this  enzyme  glucase. 

Cusenier,  by  soaking  ground  maize  in  water  at  a  tern- 

208 


MALTASE.  209 

perature  of  500,  found  that  a  great  part  of  the  amylaceous 
matter  passed  into  solution,  and  that  the  rotatory  power  of 
the  saccharine  liquid  decreased  as  the  soaking  was  pro- 
longed. This  observation  led  to  researches  in  regard  to  the 
nature  of  the  sugar  formed,  as  well  as  that  of  the  agent  pro- 
ducing this  transformation. 

A  series  of  experiments  undertaken  with  this  aim  resulted 
in  finding  that  the  maize  contains  a  special  ferment  which 
acts  on  starch,  giving  glucose  and  dextrins,  which  in  the  end 
are  themselves  transformed  into  dextrose. 

The  optimum  temperature  of  this  enzyme  is  6o°  ;  its  tem- 
perature of  destruction,  about  700. 

This  enzyme  acts  likewise  on  maltose  and  transforms  it 
into  glucose. 

Its  presence  has  been  observed  in  almost  all  the  cereals, 
but  in  a  much  smaller  quantity  than  in  maize.  There  exists 
in  the  latter  an  amount  of  glucase  which  is  more  than  enough 
to  transform  into  glucose  all  the  starch  it  contains. 

According  to  Gedulde,  it  is  possible  to  isolate  the  glucase 
of  maize  by  soaking  a  grist  with  water  and  then  precipitating 
the  filtered  liquid  with  alcohol.  The  product  obtained,  and 
dried  in  vacuo,  is  a  brownish  mass  which  is  friable  and  has  the 
following  properties: 

It  contains  about  8  to  12  per  cent  of  nitrogen.  It  gives 
the  reaction  of  guaiacum  and  hydrogen  peroxide.  Pre- 
cipitated by  alcohol,  it  is  redissolved  in  water  with  difficulty. 

It  possesses  a  relatively  weak  activity:  with  one  part  of 
precipitated  active  substance  only  100  parts  of  maltose  are 
transformed  into  glucose. 

Its  optimum  temperature  is  from  560  to  6o°.  Above 
6o°  there  is  found  a  perceptible  slackening  in  the  hydration 
it  produces.     Above  700  glucase  is  without  action. 

This  enzyme  acts  more  energetically  on  the  products  of 
decomposition  of  starch  than  on  the  starch  itself. 

According  to  Beijerinck,  glucase  can  easily  be  prepared 
from  maize  which  is  hulled  and  deprived  of  its  oil.     The  fol- 


2io  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

lowing  method  is  employed:  Three  kilograms  and  a  half  of 
maize  thus  prepared  are  treated  with  5  litres  of  water,  with 
the  addition  of  500  cubic  centimetres  of  96%  alcohol  and  of  2 
grams  of  tartaric  acid.  This  mixture  is  kept  for  30  hours 
at  150  or  20°,  then  filtered.  Thus  4-J  litres  of  a  clear  liquid 
are  obtained  in  which  is  produced  a  partial  precipitation  by 
adding  to  it  an  equal  volume  of  96%  alcohol.  The  deposit 
thus  procured  is  treated  with  acidulated  water  in  the  pro- 
portion of  0.4  grams  of  tartaric  acid  per  litre,  then  a  little 
alcohol  is  added.  The  precipitate  is  partially  redissolved  in 
the  liquid  and  the  insoluble  part  is  collected  on  a  filter.  This, 
insoluble  product  is  very  rich  in  glucase  according  to 
Beijerinck.     It  contains  1.11%  of  nitrogen. 

Other  products  may  be  obtained  by  adding  alcohol  to  the 
filtered  liquid.  The  precipitates  which  still  contain  a  certain 
quantity  of  diastases  in  solution  are  collected.  But  the  pre- 
cipitates obtained  by  these  treatments,  while  showing  a  nitro- 
gen content  of  4.78  and  2.20  per  cent,  are  less  active  than  the 
insoluble  part  spoken  of  before.  The  glucase  obtained  by 
Beijerinck  is  not,  however,  an  absolutely  pure  product,  as  he 
himself  found.     Its  impurities  must  be  due  to  mucilages. 

According  to  Beijerinck,  glucase  acts  on  maltose,  on 
starch,  and  on  dextrins,  but  more  actively  on  maltose  than  on 
dextrins;  it  causes  the  transformation  of  starch  with  much 
difficulty. 

According  to  Gonnerman,  glucase  or  an  analogous  fer- 
ment exists  in  beets  frozen  or  in  germination.  Dubourg 
and  Rhomann  have  discovered  its  presence  in  the  blood.  It 
is  also  found  in  the  urine  and  in  yeasts,  as  well  as  in  a  great 
number  of  moulds. 

The  secretion  of  maltase  by  yeast  is  of  particular  in- 
terest. 

Maltose  has  been  considered  for  a  long  time  as  a  directly 
fermentable  sugar  and,  in  reality,  during  fermentation  it  is 
impossible  to  tell  when  the  maltose  is  transformed  into 
glucose.     This  is  why  the  fermentation  of  maltose  has  been 


MALTASE.  2H 

regarded  as  an  intracellular  transformation  in  which  the  solu- 
ble ferment  has  no  part. 

Bourquelot,  Lintner,  and  Emil  Fischer  have  studied  the 
question  very  closely  and  established  the  fact  that  yeast 
always  contains  a  certain  quantity  of  maltase  which  is  re- 
tained in  the  cells  and  which  is  diffused  with  difficulty  into 
the  surrounding  liquid. 

To  extract  the  enzymes  of  yeasts,  the  cells  must  be 
crushed  with  pumice-stone  or  pounded  glass  and  the  mass 
then  soaked  in  water.  One  may  also  have  recourse  to  an- 
other means  which  seems  more  expeditious:  fresh  yeast  is 
spread  in  a  very  thin  layer ;  it  is  slowly  dried  at  400  and  then 
soaked  in  water.  Under  these  conditions,  the  maltase  of 
the  yeast  becomes  soluble.  The  enzyme  extracted  from 
the  yeasts  by  this  method  differs  in  many  respects  from  the 
active  substance  of  the  maize  which  hydrates  the  maltose. 

According  to  Gedulde,  the  glucase  of  maize  can  be  pre- 
cipitated from  its  solution  in  the  active  state  by  alcohol; 
the  maltase  of  yeast,  on  the  contrary,  is  almost  completely 
destroyed  by  this  reagent. 

Maltases  of  different  origin  also  posses*  a  very  different 
sensitiveness  towards  heat.  Lintner,  by  exposing  to  dif- 
ferent temperatures  3  specimens  of  a  solution  of  maltose,  to 
which  had  been  added  the  same  quantity  of  glucase  ex- 
tracted from  yeasts,  obtained  quantities  of  glucose  varying 
according  to  the  temperature  at  which  the  action  took  place. 

Temperature.  q{  ^AcXn.  GluC°SC  formed' 

35°. 2  hours 2.90  gr. 

40   "        3-°9 

45    "        2-°8 

The  optimum  temperature,  according  to  these  experi- 
ments, would  be  40°,  while  Cusenier's  glucase  possesses  art 
optimum  temperature  of  56°  to  6o°. 

By  trying  yeast  maltase  at  temperatures  of'400  and  500 


2  12  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

Lintner  found  the  formation  after  two  hours  of  action  of  the 
following  quantities  of  glucose: 

Temperature.  Glucose  formed. 

400 1.8 

5o° 0.3 

Therefore  the  temperature  of  500  almost  entirely  de- 
stroys the  maltase  of  yeast,  while  if  glucase  of  maize  is  used, 
the  maximum  result  is  not  reached  at  that  temperature. 

A  great  number  of  moulds  possess  the  property  of  trans- 
forming starch  into  sugar. 

By  studying  the  action  of  Aspergillus  orizce,  Atkinson 
was  the  first  to  find  that  the  diastase  of  this  mould  brings 
about  the  transformation  of  amylaceous  materials  in  a  dif- 
ferent way  from  that  of  the  diastase  of  malt.  The  final  prod- 
uct of  this  reaction  of  Aspergillus  orizcc  is  dextrose  and  not 
maltose. 

Since  then  the  same  fact  has  been  ascertained  by  Bodin 
and  Rolants  for  Amylomyces  Rouxii,  then  by  Bourquelot  and 
Laborde  for  Aspergillus  rtiger,  Penicillium  glaucum  and  Euro- 
tiopsis  Gayoni.  There  is  every  reason  for  believing  that  it 
is  a  general  fact  and  that  many  other  moulds  render  starch 
assimilable  by  the  aid  of  the  glucases  they  secrete.  Diastatic 
liquids  obtained  by  soaking  moulds  act  on  starch,  dextrins, 
and  maltose,  forming  glucose. 

According  to  Atkinson,  the  transformation  of  starch  by 
Aspergillus  orizcc  is  done  by  successive  hydrations  of  the 
molecules,  with  formation  of  maltose  as  an  intermediate 
product,  but  the  analyses  cited  for  the  support  of  this 
opinion  are  not  at  all  conclusive.  Laborde,  on  the  contrary, 
found  that  there  is  a  direct  transformation  without  the  for- 
mation of  maltose.  He  has  found  the  same  facts  in  the  case 
of  Aspergillus  niger,  Penicillium  glaucum  and  Eurotiopsis 
Gayoni. 

To  demonstrate  the  presence  of  glucase  in  an  active 
liquid,  a  2  per  cent  solution  of  maltose  is  added  to  a  certain 


MALTASE.  213 

quantity  of  the  liquid  under  examination.  A  trace  of  chlo- 
roform or  thymol  is  added  and  the  solution  is  left  at  a  tem- 
perature of  450  for  24  hours.  By  examining  the  rotation  of 
the  liquid  before  and  after  the  experiment,  one  can  easily 
follow  the  course  of  the  hydration. 

The  rotatory  power  of  the  maltose  is  (a)d  -f-  138.4  and 
that  of  glucose  52.4 

The  glucase  of  moulds  acts  more  strongly  on  starch  than 
on  maltose. 

The  formation  as  well  as  the  diffusion  of  glucases  of 
moulds  take  place  under  the  same  conditions  as  secretion 
of  sucrase  by  Aspergillus  n'iger.  During  the  development  of 
the  plant,  the  quantity  of  glucase  increases  as  the  nutritive 
substances  diminish  and  the  maximum  amount  of  enzyme 
appears  in  the  plant  at  the  moment  when  it  begins  to  utilize 
the  reserve  substances.  Thus  the  glucase  produced  in  the 
moulds  is  retained  inside  the  cells  and  diffuses  with  great 
difficulty  until  the  nutritive-medium  begins  to  be  exhausted. 

According  to  Pfeffer  and  Katz,  the  addition  of  sugar  to 
the  culture  medium  generally  diminishes  the  production  of 
glucase,  but  it  is  observedthat  the  different  kinds  of  moulds 
are  more  or  less  sensitive  to  this  action.  Thus  Penicillium 
glaucum  does  not  secrete  glucase  when  there  is  10  per  cent  of 
saccharose,  while  in  an  amount  of  30  per  cent  this  sugar  does 
not  entirely  stop  its  secretion  by  Aspergillus  niger.  Glucose 
acts  in  the  same  way  as  saccharose. 

The  presence  of  maltose  in  the  nutritive  medium  influ- 
ences the  secretion  in  a  less  degree.  Penicillium  glaucum  still 
produces  glucase  in  a  medium  containing  10  per  cent  of 
sugar. 

Nitrogenous  food  also  has  a  great  influence  on  the  pro- 
duction of  glucase.  The  well-nourished  cells  yield  the 
enzyme  in  largest  quantity. 

Moulds  may  take  nitrogen  from  very  different  sources. 
Thus  alkaline  nitrates,  peptone,  casein,  and  urea  are  equally 
favorable  to  the  cultivation  of  Eurotiopsis  and  furnish  practi- 


214  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

cally  the  same  quantity  of  glucase.  Sulphate  and  chloride  of 
ammonium,  however,  furnish  decidedly  less,  and  these  sub- 
stances act  very  unfavorably  from  the  point  of  view  of  the 
formation  of  diastase. 

According  to  PfefTer  and  Katz,  the  secretion  of  glucase 
by  Penicillium  glaucum  and  Aspergillus  niger  is  limited  by  the 
quantity  of  enzyme  already  present  in  the  nutritive  medium. 
By  removing  the  glucase  from  the  medium  with  tannin, 
they  have  found  a  more  abundant  secretion  of  diastase.  It 
is,  however,  not  very  probable  that  taking  away  the  active 
substance  can  cause  a  further  formation  of  diastase.  It  is 
more  plausible  to  think  that  in  precipitating  the  diastase  by 
tannin,  one  favors  the  diffusion  of  the  active  substances  al- 
ready formed  but  retained  by  the  cells. 

Of  all  the  moulds  studied  from  the  point  of  view  of  their 
action  on  amylaceous  materials,  Aspergillus  orizce  is  the  most 
active,  and  it  is  this  which  really  secretes  the  greatest  quan- 
tity of  maltase. 

Mucor  altcrnens  and  Amylomyccs  Rou.vii  both  belong  to 
the  class  of  moulds  which  is  rich  in  maltose.  Aspergillus 
niger  and  Penicillium  glaucum  possess  a  diastatic  power  which 
is  much  weaker,  and  Eurotiopsis  occupies  the  last  place  as  re- 
gards the  secretion  of  glucase 

According  to  Laborde,  the  saccharifying  diastases  of  As- 
pergillus niger,  Penicillium  glaucum  and  Eurotiopsis  Gayoni, 
and  which  he  designates  under  the  name  of  amylo-maltases, 
have  different  characteristics.  Starting  with  this  observa- 
tion, Laborde  conceives  the  existence  of  3  different  diastases 
having  common  characteristics  but  distinguished  by  their 
sensitiveness  towards  physical  and  chemical  agents,  as  well 
as  by  the  intensity  of  their  action. 

By  allowing  enzymes  of  the  three  moulds  to  act,  under 
the  same  conditions  on  2  per  cent  starch  paste,  Laborde 
found  perceptible  differences  for  the  3  diastases,  which  are 
given  in  the  following  table : 


MALTASE. 


"5 


Origin  of  the  Diastatic  Liquids. 


Aspergillus  niger. 


Penicillium  elaucum. 


Eurotiopsis  Gayoni. 


Duration  in 

Hours  of  the 

Action. 


96 


12 

48 
96 

12 
48 
96 


Folariscopic 
Rotation  of 

Glucose. 

the  Liquids. 

% 

gr. 

17-5 

i-3i 

14.0 

1. 61 

14.0 

1.66 

12.5 

I.3I 

12.0 

1. 61 

12.0 

1.72 

7.0 

0.S0 

9.0 

1. 61 

9-3 

1.92 

Dextrins. 


gr. 
O.56 
O.3I 
O.30 

0.31 
0.2I 
O.18 

O.16 
O.06 
O.OO 


The  difference  in  the  course  of  hydration  is  especially- 
shown  by  the  rotatory  powers  of  the  liquids  as  well  as  by  the 
relation  of  the  quantity  of  maltose  and  that  of  dextrins. 

For  1.60  of  glucose  formed  with  the  three  active  liquids, 
there  are  found  noticeably  different  quantities  of  dextrins  as 
well  as  different  rotations.  These  differences  probably  come 
from  the  fact  that  the  starch  is  not  liquefied  with  the  same 
facility  by  diastases  of  different  origin. 

Maltases  of  different  moulds  are  further  differentiated  by 
their  optimum  temperatures  as  well  as  by  their  temperatures 
of  destruction: 

Temperature        Temperature 
Optimum.         of  Destruction. 

Aspergillus  niger 6o°  8o° 

Penicillium  glaucum .45  70 

Eurotiopsis  Gayoni. 50  75 

As  regards  the  action  of  heat,  the  maltase  of  Aspergillus 
niger  approaches  the  maltase  of  maize,  while  the  active  sub- 
stance of  Eurotiopsis  Gayoni  resembles  rather  the  diastase  of 
Penicillium  glaucum  and  the  yeasts. 

There  is  also  found  another  difference  between  the  mal- 
tase of  cereals  and  the  ferments  of  moulds.  While  the  first 
acts  with  more  difficulty  on  starch  than  on  maltose,  the 
second  acts  more  vigorously  on  starch  than  on  the  products 
of  hydration. 


216  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

Moreover,  these  differences  between  the  manner  of  ac- 
tion of  diastases  are  apparent  rather  than  real.  An  extract 
of  maize  acts  energetically  in  the  cold  on  starch  and  on  mal- 
tose. But  if,  on  the  other  hand,  one  precipitates  the  active 
substance  of  an  infusion  of  maize  with  alcohol,  one  obtains  a 
product  which  hardly  acts  at  all  on  starch,  but  which,  on  the 
contrary,  acts  very  strongly  on  maltose. 

This  difference  evidently  comes  from  the  change  of 
medium.  In  the  first  case  the  action  is  due  to  the  diastase 
accompanied  by  foreign  substances  which  influence  its  ac- 
tion. In  the  second,  it  is  the  effect  of  the  diastase  alone,  or 
of  this  diastase  accompanied  only  by  substances  exercising  a 
very  slight  influence  on  the  transformation. 

It  must  then  be  admitted  that  all  the  differences  which 
we  have  found  in  maltases  of  different  origin  comes  exclu- 
sively from  foreign  substances  which  accompany  them  and 
influence  their  sensitiveness  towards  reagents. 

Most  moulds  secreting  maltases  develop  very  easily  in 
the  mashes  of  the  brewery  and  the  distillery,  as  well  as  in 
yeast-water  to  which  carbohydrates  have  been  added. 

Sanguinetti  made  a  comparative  study  of  Aspergillus 
crizce,  Mucor  alternans  and  Amylomyces  Rouxii  as  regards  sac- 
charifying and  oxidizing  powers.  He  cultivated  them  in 
mashes  containing  starch,  dextrin  or  other  carbohydrates, 
and  observed  the  progress  of  diastatic  secretions  as  well  as 
the  influence  of  nutrition  on  these  secretions. 

Here  are  some  of  his  experiments: 

In  a  flask  of  a  capacity  of  1,500  cubic  centimetres  he 
placed  500  cubic  centimetres  of  yeast-water  and  added  15 
grams  of  starch  or  dextrin. 

He  sterilized  the  liquid,  and  after  cooling  it,  sowed  dif- 
ferent flasks  at  a  temperature  of  300  with  the  spores  of  dif- 
ferent moulds. 

He  allowed  the  plants  to  develop  for  10  days,  and 
shook  the  flasks  twice  a  day  to  prevent  the  formation  of 


MAL  TASE. 


217 


spores.     He    then    determined    the    weight    of    the    plants 
formed  and  determined  the  sugar  and  alcohol  in  the  liquid. 

The  results  of  experiments  made  with  yeast-water  and 
starch  are  here  given : 


Weight  of  plant 

Dry  extract  at  ioo" 

Total  acidity,  H2S04 

Weight  of  alcohol 

Reducing  sugar (as  glucose) 

Total  reducing  sugar  after  saccha- 

rification  by  hydrochloric  acid.  .  . 

Loss  of  alcohol  per  100  of  starch  . . 


Control. 


19.00 
O.127 


16.67 


Aspergillus 
orizae. 


gr. 

2.081 

5-20 

0.670 

2.77 

I.30 


Mucor 
alternans. 


2.25 


O.667 
6.27 
O.980 
1.58 
Traces 

2.99 

41// 


Amylo- 
myces. 


gr. 
2.080 
4- 50 
O.660 
3-96 
Traces 


3-75 
25. 7# 


When  the  quantity  of  carbohydrate  which  has  disap- 
peared is  compared  with  the  quantity  of  alcohol  formed,  it 
is  found  that  there  is  obtained: 

With  Aspergillus  oriza for  14.42  of  sugar  transformed,  2.77  alcohol. 

Mucor  alternans "     13.68   "        "  "  1.58 

Amylomyces  Rouxii "     12.92   "         "  "  3.06  " 

Aspergillus  orizee  shows  itself  the  most  active  and  leaves 
in  the  15  per  cent  solution  of  starch  .85  gr.  of  untransformed 
dextrin,  while  the  Mucor  alternans  and  Amylomyees  furnish 
twice  as  much  unattacked  dextrin. 

When  in  these  experiments  starch  is  replaced  by  dextrins 
a  still  less  complete  saccharification  is  observed. 

After  10  days  of  development  there  is  still  found  3.80  of 
dextrins. 

If  the  time  of  the  action  and  the  weight  of  the  plant  are 
taken  into  consideration,  it  is  seen  that  the  quantity  of  active 
substance  secreted  by  the  moulds  is  relatively  very  slight, 
even  with  Aspergillus  orizee;  2.081  gr.  of  plant  do  not  suf- 
fice, as  we  have  just  seen,  to  transform  15  grams  of  starch. 
while  .5  gr.  of  malt  produce,  under  similar  conditions,  a  com- 
plete transformation. 

The  maltase  of  fungi  is  very  sensitive  to  the  medium, 
fiodin  and  Rolants  have  studied  the  action  of  oxygen  and  of 


2i; 


THE  ENZYMES  AND    THEIR  APPLICATIONS. 


the  acidity  of  the  medium.  The  following  experiment  af- 
fords some  data  on  this  subject: 

Bring  a  distillery  residue  to  different  degrees  of  acidity 
and,  in  the  sterilized  liquid,  cultivate  Amylomyccs  under  dif- 
ferent conditions. 

In  one  culture  let  the  plant  develop  at  the  surface  of  the 
liquid  (culture  S) ;  in  another  let  the  development  take  place 
deep  down  (culture  P) ;  in  a  third  (culture  A)  let  a  current  of 
air  pass  for  48  hours. 

The  following  results  were  obtained  after  a  fermentation 
of  4  days  at  a  temperature  of  260 : 


Alcohol,  per  litre 

Acidity 

Reducing  sugar  in  glucose 

Total  sugar ■ 

Weight  of  amylomyces  obtained 
in  a  pressed  state 


Neutral  Residue. 


3-4C.C. 
O.36 

4-83 
10.23 

10.23 


5.5CC. 
0.83 
2-33 

7-3 

4.6gr. 


3  c.c. 
0.4 
1.64 

5-57 

8.15  gr 


Residue  with  Acidity 
equivalent  to  3.4  gr.  of 
Sulphuric  Acid. 


3  C.C. 
2.69 

4-31 

17.71 

17.71 


i.Sc.c 
3-13 

3-5 
17.71 

o.25gr 


1.7C.C. 
2.69 

3-4 
13-28 

2.30gr 


It  is  clear  that  aeration  is  very  favorable  to  the  develop- 
ment of  the  plant,  since  there  are  8.15  gr.  of  plants  in  the 
aerated  liquid,  while  a  culture  from  which  the  air  was  ex- 
cluded furnishes  only  4.6  gr. 

The  quantity  of  acid  formed  during  the  development  is  in 
direct  proportion  with  the  initial  acidity  and  is  smaller  ac- 
cording as  the  acidity  of  the  medium  at  the  beginning  is 
stronger. 

Maltase  acts  very  well  in  a  slightly  acid  medium,  but  its 
action  is  stopped  by  an  amount  of  organic  acid  correspond- 
ing to  2  grams  per  litre  of  sulphuric  acid. 

BIBLIOGRAPHY. 

Dubourg. — Recherches  sur  l'amylase  de  l'urine.     These,  Paris,  1889. 
Bourquelot. — Recherches   sur   les   proprietes   physiologique   du   maltose. 
Comptes  Rendus,  1883. 


MALTASE.  219 

Cusenier. — Sur  une  nouvelle  matiere  sucree  diastasique  et  sa  fabrication. 

Monit.  scientif.,   1886,  p.  718. 
Brown  and   Heron. — Ueber  die  hydrolitischen  Wirkungen  des  Pancreas 

und  des  Diinndarms.     An.  chim.  et  pharm.,   1880,  228. 
Lintner  und  Kroeber. — Verschiedenheit  der  Hefeglucase  von  Maisglucase 

und  Invertin.     Ber.  der  deutsch,  chem.  Gesellsch.,  1895,  p.  1050. 
G.  H.    Morris. — Hydrolyse   de  la   maltase   par   la  levure.     Proc.    Chem. 

Soc,  1895. 
Laborde. — Recherches  physiol.  sur  une  moisissure  d'Eurotiopsis  Gayoni. 

Ann.  de  l'lnst.  Pasteur,  1898. 
Sanguinetti. — Contrib.  a  l'etude  de  l'amylomyces  Rouxii.    Ann.  de  l'lnst. 

Pasteur,  1897. 
Bodin   et   Rolants. — Contrib.    a   l'etude   de   l'utilisation   de   l'amylomyces 

Rouxii.     Biere   et   boissons   fermentees.     Mars.    1897. 
Pfeffer  und  Katz. — Schriften  der  konig.  sach.  Gesellschaft  der  Wissen- 

schaft.     Leip.,   1896. 
Fischer  und  Lindner. — Enzyme  von  Schizosaccharomyces  octosporus  und 

Saccharomyces  Marxii.     Berichte  der  deutschen  chemischen  Gesell- 
schaft, 1895,  I,  p.  984. 
Beijerinck. — Centralblatt  fur  Bakteriologie,   n.   Abth.,  2.  Jahrg.,   1898. 
Wroblewsky. — Ueber  die   chemische    Beschaffenheit   der   amylolytischen 

Fermente.     Berichte   der  deutsch.   chem.    Gesellschaft,    1898. 


CHAPTER  XVIII. 

INDUSTRIAL  APPLICATIONS  OF  MALTASE. 
CEREALOSE. 

Industrial  Manufacture  of  Glucose  by  Enzymes. — Cuse- 
nier,  having  found  glucase  in  maize,  sought  to  apply  this  dis- 
covery to  the  glucose  industry.  By  replacing,  in  this  manu- 
facture, acid  by  enzymes  contained  in  the  grains,  he  suc- 
ceeded in  making  a  product  of  great  value  which  is  found  in 
commerce  under  the  name  of  cerealose. 

Cerealose  is  obtained  in  the  form  of  a  crystalline  mass 
containing  maltose  and  glucose. 

The  manufacture  of  cerealose  is  as  yet  little  developed,, 
for  the  process  of  Cusenier  leaves  much  to  be  desired  from 
the  point  of  view  of  yield  and  cost  of  production,  which  is. 
greater  than  that  of  the  glucose  obtained  with  acids. 

The  difficulties  encountered  in  this  industry  are  various. 

The  maize  contains  hydrating  ferments  in  a  quantity 
more  than  sufficient  to  transform  all  the  starch  into  sugar, 
but  practically  it  is  very  difficult  to  have  conditions  favorable 
to  the  action  of  these  ferments.  It  results  that  the  transfor- 
mation is  far  from  complete. 

When  ground  maize  is  soaked  with  4  volumes  of  water  at 
a  temperature  of  6o°  for  24  hours,  60  to  65  per  cent  of  amy- 
laceous materials  is  extracted  in  the  form  of  sugar.  A  more 
prolonged  soaking  does  not  bring  about  a  perceptibly  bet- 
ter result.  The  enzyme  acts  only  on  a  part  of  the  starch 
and  unattacked  grains  of  starch  always  remain,  although  the 
saccharine  solution  obtained  is  very  rich  in  active  substances. 
It  is  this  peculiarity  in  particular  which  makes  the  manufac- 

220 


INDUSTRIAL   APPLICATIONS   OF  MALTASE.  221 

ture   of   glucose   by   glucase   difficult.     The   operation   can 
really  never  be  carried  to  perfection. 

One  is  obliged  to  have  recourse  to  a  continuous  method 
of  work. 

The  method  of  procedure  is  here  outlined : 
Five  hundred  kilograms  of  coarsely  ground  maize  are 
poured  into  an  apparatus  furnished  with  a  double  wall  and  a 
stirrer.  Twenty  hectolitres  of  water  at  650  are  added.  The 
temperature  is  kept  at  58°-6o°  for  6  to  8  hours,  the  stirrer 
being  kept  in  motion. 

The  transformation  of  amylaceous  materials  into  glucose 
is  followed  by  the  observation  of  the  density  and  rotatory 
power  of  the  mash.  During  all  the  course  of  the  operation, 
the  density  of  the  liquids  increases,  while  the  rotatory  power 
diminishes.  The  operation  is  considered  as  finished  when 
the  mash  containing  10  per  cent  of  dry  materials  marks  400 
to  450  on  the  Soleil  polarimeter.  Then  the  saccharine  solu- 
tion is  separated  by  filtration  from  the  extracted  grain,  which 
still  contains  quite  considerable  quantities  of  unattacked 
starch.  The  juice  is  decolorized  with  bone-black  and  evap- 
orated in  a  vacuum  to  a  concentration  of  400  to  42  °  Baume, 
after  which  the  syrup  is  placed  in  vessels,  where  it  solidifies 
at  once  after  it  has  been  primed  with  crystallized  glucose. 

The  starch  not  attacked  in  the  first  operation  is  subjected 
to  further  treatment.  The  grist  of  maize  remaining  on 
the  filter  is  submitted  to  cooking  under  slight  pressure. 
The  starch  obtained  by  this  operation  is  saccharified  at  a 
temperature  of  630,  by  the  aid  of  a  small  quantity  of  malt 
(1  to  2  per  cent),  and  the  dextrinated  mashes  replace  water 
in  the  following  operation.  With  the  malts  coming  from 
500  kilograms  of  maize,  nearly  20  hectolitres  of  dextrinated 
mash  is  obtained.  Into  this  mash  are  introduced  400  kilo- 
grams of  ground  maize  and  saccharification  is  allowed  to  pro- 
ceed for  6  to  8  hours  at  a  temperature  of  58°-6o°. 

The  maltase  contained  in  the  barley  malt  can  also  be 
utilized  for  the  manufacture  of  glucose. 


222  THE   ENZYMES  AND    THEIR  APPLICATIONS. 

The  infusion  of  malt  has  very  little  action  on  the  maltose, 
but  the  crushed  malt  acts  vigorously  on  the  maltose  syrups, 
which  are  transformed  into  dextrose  syrups.  For  this  kind 
of  work  it  is  well  to  put  the  malt  in  contact  with  the  syrups  at 
20°-25°  Baume  and  not  with  diluted  juices. 

In  saccharified  and  very  concentrated  mashes,  the  extrac- 
tion of  the  diastases  of  the  malt  is  made  more  easily  than  in 
syrups  of  low  concentration. 

Cerealose  has  the  following  average  composition : 

Maltose 2.5% 

Glucose 72     " 

Dextrin 2.5  " 

Water 20     " 


CHAPTER  XIX 

INDUSTRIAL  APPLICATIONS  OF  MALTASE.— (Continued.) 

JAPANESE  AND  CHINESE  YEASTS. 

Manufacture  of  Japanese  yeast. — Preparation  of  koji. — Changes  produced 
in  the  rice. — Composition  of  koji. — Action  of  salts. — Manufacture  of 
"  moto  "  leaven. — Manufacture  of  "  sake  "  beer. — Composition  of  moto. 
— Composition  of  sake. — Manufacture  of  Chinese  yeast. — Prop- 
erties of  Chinese  yeast. — Influence  of  temperature  and  chemical 
agents. — Oriental  methods  of  distillation. — Utilization  of  Oriental 
processes  in  the  distilleries  of  Western  countries. — Works  of  Taka- 
mine,  Collette,  and  Boidin. 

Japarjese  Yeast. — In  certain  countries  of  the  far  East  al- 
coholic beverages  are  manufactured  with  amylaceous  ma- 
terials. In  Japan  a  kind  of  beer  is  made  which  is  called 
sake.  In  China  and  Cochin-China  a  brandy  is  prepared  from 
rice,  "  choum-choum,"  which  contains  from  34  to  42% 
of  alcohol. 

The  methods  employed  by  the  Orientals  differ  radically 
from  European  methods.  The  saccharification  of  the  amy- 
laceous materials  and  the  fermentation  are  brought  about  by 
special  ferments  which  are  cultivated  as  an  industry. 

The  active  agent  which  the  Japanese  use  is  called  koji. 
The  ferment  which  serves  for  the  manufacture  of  Cochin- 
China  brandy  is  called  niigcn  or  men. 

Chinese  and  Japanese  yeasts  owe  their  activity  to  moulds 
which  secrete  maltase  and  probably  zymase. 

In  the  Chinese  yeast,  the  predominating  organism  is 
Am\lomyccs  Rouxii.     Koji  owes  its  activity  to  Eurotium  orizce. 

Korschelt  and  Atkinson  published  the  first  data  on  the 
preparation  and  utilization  of  Japanese  yeasts. 

223 


224  THE  ENZYMES  AND   THEIR  APPLICATIONS. 

Preparation  of  Koji. — The  grains  of  rice  destined  for 
the  manufacture  of  koji  are  first  cleansed  and  beaten  so 
as  to  free  them  of  their  covering,  then  they  are  submitted  to 
a  soaking  of  a  dozen  hours.  The  grains  are  then  cooked  in 
a  current  of  steam  until  they  have  reached  a  certain  consist- 
ency, then  are  spread  on  mats,  which  are  vigorously  shaken 
to  prevent  the  grains  from  uniting  in  lumps.  The  rice  is 
then  sown  with  the  spores  of  a  mould,  Eurotium  orizcE. 
The  spores  of  this  mould,  which  form  a  commercial  product* 
in  Japan,  are  mixed  with  the  rice  in  the  proportion  of  I  part 
of  spores  to  40,000  parts  of  rice.  They  are  distributed 
throughout  the  mass  by  a  vigorous  shaking  of  the  mat  and 
the  whole  is  taken  to  the  malt-house. 

Atkinson,  professor  at  the  University  of  Tokio,  to  whom 
we  owe  the  description  of  this  industry,  describes  the  special 
construction  of  these  malt-houses.  They  are  long  subter- 
ranean passages,  joining  each  other,  4  to  10  metres  long, 
from  2.10  m.  to  2.40  m.  wide  and  1.20  m.  high.  These  malt- 
houses  are  never  heated  except  at  the  beginning  of  the  cold 
season.  The  rice,  mixed  with  spores,  is  heaped  up  in  the 
malt-house,  covered  with  mats  and  left  in  this  state  over 
night. 

On  the  second  day  it  is  sprinkled  with  a  certain  quantity 
of  water,  if  it  is  not  to  be  used  for  the  manufacture  of  the 
beer  called  sake. 

The  koji  is  then  spread  in  a  very  thin  layer  and  left.  The 
third  day  the  rice  is  again  heaped  up  for  about  4  hours.  At 
the  end  of  this  time  the  grains  are  covered  with  a  light  fleece 
coming  from  the  mycelium  of  the  mould.  The  rice  is  then 
cooled  by  shaking,  and  disposed  in  thin  layers  on  mats,  to 
which  is  given  a  lateral  motion  to  prevent  the  formation  of 
lumps. 

Under  these  conditions,  the  vegetation  develops ;  the 
mycelium  winds  about  among  the  grains  and  the  fourth  day 
the  koji  forms  a  kind  of  cake  which  is  all  ready  to  be  used. 

Koji  is  used  in  different  branches  of  Japanese  industry;  in 


INDUSTRIAL   APPLICATION  OF  MALTASE.  225 

the  making  of  bread,  in  the  preparation  of  the  "  soy  "  sauce; 
but  especially  in  the  brewery  for  the  preparation  of  sake. 

Temperature  of  Germination. — In  Atkinson's  account  are 
found  some  data  on  the  variations  of  temperature  in  the 
course  of  this  manufacture.  When  after  soaking,  the  grains 
of  rice  have  been  dried  by  shaking  the  mats,  they  have  a  tem- 
perature of  28°-30°.  The  second  day,  after  being  scattered, 
the  temperature  falls  to  23°-26°,  to  rise  again  afterwards  in 
the  malt-house  to  300.  The  next  day,  the  third,  Atkinson 
observed  it  to  reach  40°-4i°.  The  rice  is  then  cooled,  but  it 
heats  again  to  370.  These  figures  are,  however,  only  ap- 
proximate ;  they  change  according  to  the  time  of  year.  In 
the  month  of  May  the  temperature  of  the  malt-house  has 
been  observed  to  be  from  24°-26° ;  the  temperature  of  the 
koji  was  then  from  25°-26°.  In  the  month  of  December  the 
thermometer  marked  2j°  in  the  malt-house  and  the  rice 
showed  a  temperature  of  390.  This  increase  in  temperature 
is  explained  by  very  vigorous  oxidation  produced  by  the 
moulds :  often  a  difference  of  10  degrees  is  found  between 
the  temperature  of  the  koji  and  the  external  temperature. 
At  a  temperature  of  400  considerable  losses  of  starch  must 
occur,  as  well  as  a  perceptible  change  in  the  diastase 
secreted  by  the  moulds. 

According  to  more  recent  data  on  the  preparation  of  koji, 
the  Japanese  manufacturers  take  pains  not  to  exceed  a  tem- 
perature of  250.  The  whole  duration  of  the  manufacture 
from  the  moment  of  sowing  to  that  of  the  complete  develop- 
ment of  the  plants  is  only  three  days. 

Changes  Produced  in  the  Rice. — The  transformation  of 
the  rice  into  koji  appears  then  like  a  true  phenomenon  of 
oxidation.  In  fact,  the  rice  used  in  this  process  loses  as 
much  as  1 1  per  cent  of  its  starch ;  this  carbohydrate  is  oxi- 
dized with  liberation  of  carbonic  acid  and  formation  of  water. 

The  koji  presents  the  aspect  of  a  cake  formed  of  grains  of 
rice  bound  together  by  fungus  threads.  The  grains  ex- 
tracted from  this  cake  are  covered  with  a  sort  of  down,  and 


226  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

an  incision  made  on  one  of  them  shows  that  the  exterior  cells 
are  penetrated  by  the  threads  of  the  mycelium,  while  the  in- 
terior remains  unattacked  and  even  acquires  a  certain  hard- 
ness. 

The  mould  in  the  course  of  development  attacks  the  al- 
buminoid materials  which  are  found  in  the  rice ;  these  sub- 
stances become  soluble  and  the  fermenting  power  of  the  koji 
increases  with  their  solution. 

The  composition  of  koji  dried  at  ioo°,  according  to  At- 
kinson, is  here  given : 

f  Dextrose 25.02 

r,  ,    .,     .  _,ri        1   Dextrin  (by  difference) 3.88 

Part  soluble  in  water:  37.76%...  -{   c   ,    ,  ,      v  /  J 

■"   '  j    Soluble  ash 0.52 

[  Soluble  albuminoids 8.34 

f  Insoluble  albuminoids 1.50 

j   Insoluble  ash 0.09 

Part  insoluble  in  water:  62.24$.  i   Fatty  bodies 0.45 

I   Cellulose 4.20 

(^  Starch  (by  difference) 56.00 

The  fresh  koji  contains  25.82  per  cent  of  water. 

The  growth  of  Enrotium  orizce  on  the  grain  causes  the 
proportion  of  soluble  nitrogen  in  the  latter  to  increase.  In 
dried  koji  there  is  found  a  total  of  9.84  per  cent  of  albuminoid 
materials,  the  soluble  albuminoid  materials  being  represented 
by  8.34  per  cent.  In  the  rice  not  transformed  into  koji  the 
quantity  of  soluble  albuminoid  materials  is  only  1.38  per  cent. 
There  is  also  a  difference  of  solubility  between  rice  and  koji. 
When  the  koji  has  not  been  heated  to  ioo°,  it  for  the  most 
part  dissolves.  After  a  short  contact  with  cold  water  12  or 
15  per  cent  of  its  total  weight  dissolves.  If  the  contact  with 
water  is  prolonged  the  diastase  continues  to  act  and  at  the 
end  of  a  longer  or  shorter  time  30  to  60  per  cent  of  the  koji 
enters  into  solution. 

Action  of  Salts. — The  enzyme  of  koji  is  influenced  by  the 
acidity  of  the  medium.  Lactic  acid,  in  an  amount  of  0.05  per 
cent,  is  favorable;  the  amount  of  0.1  per  cent  possesses  a  re- 
tarding action. 

The  diastase  is  equally  sensitive  to  the  action  of  sodium 
hloride.     The   influence    of   this   salt   was   determined   by 


INDUSTRIAL   APPLICATION   OF  MALTASE.  227 

Watanabe.  To  5  grams  of  dry  starch,  gelatinized  and 
cooled,  he  added  different  amounts  of  sodium  chloride  and 
then  added  to  each  of  these  specimens  the  same  quantity  of 
extract  of  koji.  He  then  left  it  at  the  ordinary  temperature 
for  1  hour,  then  brought  the  volume  up  to  250  cubic  cen- 
timetres and  filtered.  The  action  of  the  salt  is  determined 
by  the  aid  of  the  reducing  power  and  the  rotatory  power  of 
the  solution. 

Common  salt  Reducing  power  Specific  rotatory 

per  100  of  starch.         (on  copper  oxide).  power. 

o  30.8   173-8° 

10  28.6   179.3 

30  25.1  182.6 

50  23.8 187.6 

75   2°-9 190-3 

100  20.1  189.1 

150  19. 1    190.2 

200  18.0  192.2 

300   16.9  194.1 

500  14-4  197-5 

The  increase  in  rotatory  power  is  easily  seen  and  the 
diminution  in  reducing  power  as  the  amount  of  sodium 
chloride  increases. 

Manufacture  of  Moto. — Koji  is  used  in  Japanese  industry 
as  agent  of  saccharification,  fermentation,  and  the  manufac- 
ture of  the  beer  called  "  sake."  The  operations  necessary  for 
this  manufacture  are  divided  into  two  classes :  first  the  prep- 
aration of  a  strong  ferment,  called  moto,  then  the  manufac- 
ture of  the  mash  and  its  fermentation. 

For  the  manufacture  of  moto,  there  are  used  as  raw 
materials  rice  cooked  by  steam,  koji,  and  water  mixed  in  the 
following  proportions: 

Rice 68  parts. 

Koji 21       " 

Water 72       " 


2  28  THE  ENZYMES  AND    THEIR   APPLICATIONS. 

The  manufacture  of  moto  is  carried  on  in  two  stages.  In 
the  first  stage,  which  lasts  from  5  to  6  days,  the  mixture  of 
grains  and  koji  is  distributed  in  different  vessels.  Under  the 
influence  of  the  koji,  the  rice-starch  becomes  liquefied  and 
saccharified.  The  temperature  does  not  exceed  a  few  de- 
grees, and  the  fermentation  is  extremely  slow.  In  the  second 
stage,  the  combined  liquids  are  heated  to  about  25 ° ;  fermen- 
tation commences,  and  the  temperature  rises,  but  never  ex- 
ceeds 300.  The  manufacture  of  moto  lasts  from  16  to  18 
days,  and  the  mature  leavens  show  as  much  as  10  per  cent  of 
alcohol. 

The  following  table  shows  the  composition  of  moto  in  the 
first  stage  of  the  manufacture : 

After  3  days.        After  5  days. 
Per  cent.  Per  cent. 

Dextrose 7.35  12.25 

Dextrin 5.12  5.69 

Glycerin ~\ 

Ash J-  Trace  0.48 

Albumen J 

Fixed  acids 0.017  0.019 

Volatile  acids ....  0.008 

Water  by  difference 87.513  81.553 

Undissolved  starch 20.43  15A& 

Its  composition,  during  the  second  stage,  is  shown  by  the 
following  table : 

7  days.  10  days.  12  days.  14  days. 

Alcohol 5.2  8.61  9.41  0.62 

Dextrose 5.4  0.99  0.49  0.50 

Dextrin 7.0  2.81  2.72  2.57     • 

Glycerin 1.14  2.82  2.35  1.93 

Fixed  acids 0.31  0.24  0.31  0.30 

Volatile  acids 0.15  0.11  0.05  0.03 

Water  by  difference  80.80  84.42  84.67  85.47 

Undissolved  starch.  10.68  12.46  11.55  I2-05 


INDUSTRIAL   APPLICATION  OF  MALTASE.  229 

Manufacture  of  Sake. — For  the  manufacture  of  sake  the 
same  primary  materials  are  used  as  for  the  preparation  of 
moto. 

The  rice  is  saccharified  by  means  of  koji,  and  mo/o-yeast 
is  added.  Saccharification  and  fermentation  are  produced 
simultaneously.  After  a  few  days  of  slow  fermentation,  the 
mash  becomes  heated  and  begins  to  ferment  very  vigorously. 

The  mashes  of  sake  have  a  very  strong  concentration, 
reaching  35 °  Balling.  The  fermentation  of  these  mashes 
lasts  from  15  to  17  days.  Generally  the  amount  of  alcohol 
produced  is  from  12  to  13  per  cent,  and  in  some  manufac- 
tories from  14  to  15  per  cent. 

The  table  shows  the  composition  of  the  must  after  28  days 
of  fermentation : 

Alcohol !3-23 

Dextrose 

Dextrin 0.41 

Glycerin 1.99                 1 

Fixed  acids o.  107 

Volatile  acids 0.061 

Water 84.202 

Undissolved  starch 4.18 

The  fermented  mashes  are  filtered,  and  not  further  used ; 
in  certain  manufactories,  however,  the  mash  is  kept,  and 
made  to  undergo  a  secondary  fermentation. 

The  starch  contained  in  the  residues  is  used  again  after 
cooking. 

To  preserve  the  sake,  the  fermented  liquids  are  reheated 
to  50°-66°.  The  Japanese,  therefore,  adopted  a  method  of 
sterilization  before  Europe  had  any  knowledge  of  the  pro- 
cess of  Pasteurization.  As,  after  sterilization,  they  put  the 
liquid  back  into  an  unsterile  receptacle,  they  lose  a  part  of  the 
good  of  this  operation. 

Sake  differs  from  beer  by  the  small  quantity  of  dextrin 
and  dextrose  which  it  contains. 


230  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

In  the  manufacture  of  moto,  a  very  strong  alcoholic  fer- 
mentation is  developed.  The  explanations  as  to  the  cause 
of  this  fermentation  are  very  contradictory.  According  to 
certain  authors,  it  is  due  to  a  Saccharomyces  which  is 
developed  spontaneously  in  the  mash ;  others  claim  that  it  is 
due  to  a  mucor  which,  under  certain  culture  conditions,  is 
transformed  into  an  alcoholic  ferment. 

It  is  incontestable  that  Aspergillus  orizoz  as  well  as  numer- 
ous other  moulds  can  acquire  a  fermenting  power  when  cul- 
tivated under  certain  conditions ;  but  these  moulds  generally 
give  little  alcohol  and  ferment  very  slowly.  Moreover,  as. 
Saccharomyces  have  always  been  found  in  koji,  there  is 
every  reason  for  believing  that  the  fermentation  comes  prin- 
cipally from  the  yeast. 

Chinese  Yeast. — Chinese  yeast  has  been  particularly 
studied  by  Calmette,  who  published  detailed  data  on  the  work 
in  the  distilleries  of  the  far  East.  It  is  from  these  works  that 
we  obtain  the  following  information : 

Chinese  yeast  possesses  the  double  property  of  sacchari- 
fying and  causing  to  ferment  the  amylaceous  materials  with 
which  it  comes  in  contact.  It  is  found  in  commerce  in  the 
form  of  little  loaves  of  rice  which  give  out  a  musty  odor. 
These  loaves  are  filled  with  bacteria,  yeasts,  and  different 
kinds  of  moulds. 

The  active  agent  contained  in  these  loaves  is  a  mould 
which  penetrates  all  through  the  mass  by  its  mycelial  ramifi- 
cations, a  mould  called  by  Calmette  Amylomyces  Rouxii. 

The  Amylomyces  is  very  abundant  in  the  loaves  which  con- 
stitute the  Chinese  yeast.  When  this  mould  is  cultivated  in 
glucose  agar-agar,  it  develops  very  rapidly  and  forms  at  the 
end  of  48  hours  a  sort  of  veil  extending  over  the  whole  sur- 
face of  the  culture.  Potato  and  sweet  potato,  sown  with  the 
spores  of  this  mould,  become  covered  with  a  light  floury 
coating  which  at  last  becomes  transparent  and  invisible. 
The  Amylomyces  develops  normally  in  gelatine,  peptone,  and 


INDUSTRIAL   APPLICATION  OF  MALTASE.  231 

beef  broth  which  is  peptonized  and  alkaline,  although  a  slight 
acidity  is  more  favorable  for  it. 

The  mould  coagulates  milk  in  24  hours  and  reddens  it 
when  it  has  been  previously  colored  blue  by  litmus. 

As  a  general  rule  saccharine  mashes  containing  potas- 
sium phosphate  are  suitable  for  the  development  of  the  plant ; 
however,  the  media  where  it  grows  the  best  are  beer-worts, 
liquid  or  gelatinized,  and  amylaceous  substances  which  have 
been  cooked  by  steam. 

When  the  mould  is  developed  away  from  the  air,  it  takes 
on  a  fleecy  aspect  and  produces  small  quantities  of  alcohol. 
If,  on  the  contrary,  it  lives  at  the  surface  of  the  mash  or  wort, 
it  consumes  the  sugar  and  produces  oxalic  acid.  Cultivated 
in  the  air  in  a  medium  containing  dextrins  or  starch,  it  trans- 
forms cane-sugar  into  fermentable  sugar. 

Amylomyccs  is,  according  to  Calmette,  the  ferment  which 
transforms  starch  into  sugar  with  most  energy. 

Calmette,  by  following  the  growth  of  this  plant,  has  found 
that  in  contact  with  the  air  the  mycelium  forms  conidia; 
when  the  air  is  excluded  they  extend  their  hyphae  in  every 
direction  and  are  reproduced  by  direct  budding. 

This  mould  differs,  from  botanical  and  physiological 
points  of  view,  from  all  other  known  species:  it  seems  to  ap- 
proach the  trichophytes,  while  by  its  mode  of  reproduction  as 
well  as  by  its  physiological  properties  it  recalls  the  branched 
Saccharomyccs. 

Diastase  of  Chinese  Yeast. — The  diastase  contained  in 
the  cells  of  Amylomyccs  presents,  according  to  Calmette,  all 
the  characteristics  of  the  amylase  of  malt.  This  diastase  is 
secreted  by  the  hyphae. 

Calmette  also  attributes  to  the  Amylomyccs  the  property 
of  secreting  sucrase.  In  reality  the  diastase  secreted  is  glu- 
case,  and  this  enzyme  has  nothing  in  common  with  either 
amylase  or  sucrase. 

To  obtain  a  diastatic  solution  of  this  ferment,  recourse  is 


232  THE  ENZYMES   AND    THEIR   APPLICATIONS. 

had  to  a  method  similar  to  that  recommended  by  Fernbach 
for  the  preparation  of  the  sucrase  of  yeasts. 

First  the  mould  is  cultivated  in  sterilized  Raulin's 
medium,  or  better  still  in  beer-wort,  and  when  the  plant  has 
reached  its  normal  growth,  the  liquid  is  replaced  by  sterilized 
water.  After  a  stay  of  nearly  60  hours  in  a  thermostat  at 
380,  the  diastases  contained  in  the  cells  are  diffused  into  the 
surrounding  liquid ;  then  the  water  is  taken  away,  and  shows 
active  diastatic  properties. 

The  following  experiment  has  been  made  by  Calmette  to 
determine  the  activity  of  the  enzyme  of  Amylomyces.  The 
diastatic  solution  is  divided  into  several  portions  of  30  cubic 
centimetres  each,  which  are  added  to  a  1  per  cent  solution  of 
starch,  sterilized,  and  weighing  120  grams.  Each  portion  re- 
ceives a  drop  of  oil  of  garlic,  which  plays  the  part  of  anti- 
septic. The  whole  is  taken  to  the  thermostat,  where  sac- 
charification  proceeds.  The  quantities  of  sugar  obtained  are 
here  shown : 

After   1   hour 0.12  gr. 

"      6      "      0.28 

"12      "      0.33 

"24      "      0.35 

It  is  seen  that  the  proportion  between  the  duration  of  the 
action  and  the  quantity  of  product  formed  ceases  after  12 
hours ;  it  appears,  therefore,  that  the  diastase  is  altered  after 
that  length  of  time. 

To  estimate  the  fermenting  power  of  the  plant  cultivated 
on  rice,  Calmette-  suggests  the  following  method : 

One  hundred  grams  of  steamed  rice  are  sown  with  hyphse 
from  a  pure  culture  of  Amylomyces ;  it  is  left  3  days  at  a  tem- 
perature suitable  for  the  development  of  the  mould.  Then 
the  mass  is  ground  with  500  grams  of  water  and  the  whole 
poured  on  the  membrane  of  a  dialyser  floating  on  distilled 
and  sterilized  water. 

The  starch  paste  does  not  dialyse  and  the  membrane  al- 


INDUSTRIAL   APPLICATION   OF  MALTASE.  ^t, 

lows  only  the  glucose  and  the  diastases  in  solution  to  be 
transfused.  There  is  then  formed  under  the  dialyser  a  new 
diastatic  solution  in  which  the  sugar  is  measured ;  then  cer- 
tain quantities  of  these  solutions  are  stirred  in  a  i  per  cent 
solution  of  starch  paste.  After  saccharification  the  sugar 
formed  is  measured  and  the  sugar  introduced  with  the 
infusion  of  Amylomyces  is  deducted. 

The  diastase  extracted  from  fresh  cultures  produces  a 
more  intense  hydration  than  the  diastase  extracted  from  old 
cultures. 

The  filtration  of  diastatic  solutions  in  Chamberland 
bougies  takes  away  all  their  fermenting  power. 

The  method  adopted  by  Calmette  for  determining  the 
diastase  leaves  much  to  be  desired.  It  is  evident  that  by  this 
process  only  a  small  part  of  the  diastase  contained  in  the 
plants  is  obtained.  To  take  account  of  the  diastatic  power 
of  the  amylaceous  materials  on  which  moulds  have  devel- 
oped, it  is  necessary  to  crush  them,  reduce  them  to  powder 
or  paste,  and  use  the  substance  prepared  in  this  way.  For 
example,  i  gram  of  this  substance  may  be  taken,  mixed  with 
10  grams  of  gelatinized  starch  and  saccharification  allowed 
to  proceed  for  an  hour  at  400.  From  the  quantity  of  sugar 
found  must  then  be  deducted  the  sugar  formed  under  the  in- 
fluence of  the  active  materials  alone,  for  it  is  particularly  a 
question  of  determining  the  quantity  of  sugar  which  the 
gram  of  active  matter  can  give  by  itself  under  the  conditions 
of  the  experiment. 

Influence  of  Temperature  and  Chemical  Agents.— The 
temperature  most  favorable  for  the  development  of  Amy- 
lomyces is  from  35°-38°.  At  this  temperature  the  plant 
produces  the  strongest  hydration.  Above  380,  or  lower  than 
23°,  growth  weakens ;  at  72°  the  diastase  is  destroyed.  The 
plant  itself  is  destroyed  by  a  stay  of  a  half-hour  at' 75°  or  15 
minutes  at  8o°. 

The  presence  of  salts  appears  to  be  of  little  disadvantage 
to  the  diastase.      Calmette  has  determined  the  amounts  of 


234  THE  ENZYMES   AND   THEIR  APPLICATIONS. 

different  substances  which  do  not  influence  the  diastase;  he 
lias  found : 

1.10%  of  phenol. 

0.05  "  of  silver  nitrate. 

0.10  "  of  copper  sulphate. 

o.  10  "  of  iron  sulphate. 

0.10"  of  zinc  sulphate. 
Oil  of  mustard,  used  in  small  quantities,  has  no  influence 
on  the  development  of  the  plant.  Five  per  cent  of  glycerin 
produces  a  favorable  effect.  Oil  of  garlic  in  very  small  quan- 
tity and  mercuric  chloride  in  0.005  Per  cent,  on  the  contrary, 
check  the  growth  of  the  mould. 

Manufacture  of  Chinese  Yeast. — Chinese  yeast,  the  prep- 
aration of  which  demands  quite  complicated  operations,  is 
in  the  far  East  the  object  of  a  very  interesting  industry.  The 
apparatus  needed  for  this  preparation  is  quite  simple ;  being 
composed  of  mats,  shelves,  sieves,  a  granite  mortar,  and  a  cir- 
cular trough.  The  raw  materials  are  hulled  rice  and  various 
Icinds  of  aromatic  plants  which  give  a  special  perfume  to  the 
alcohol  formed  and  which,  furthermore,  undoubtedly  act  as 
antiseptics. 

These  plants  are  exceedingly  numerous;  the  best  known 
are  the  Sinapis  alba,  Caryophyllus  aromaticus,  cinnamon,  Juper 
nigrum,  cloves,  etc.  The  aromatic  plants  and  the  rice  are 
separately  ground  and  after  pulverization  mixed  and  ground 
with  water  to  form  a  soft  paste.  This  paste  is  formed  into 
little  disks  a  centimetre  thick,  which  are  placed  on  a  mat 
after  having  been  sown  with  mould  by  the  aid  of  balls  of  rice 
"which  are  introduced  into  the  paste.  The  mats  are  then  put 
on  shelves,  covered  with  straw  matting,  and  the  mould  al- 
lowed to  develop  at  a  temperature  of  280  or  300.  After  two 
days  the  moulds  have  covered  the  disks  with  a  fine  down ;  the 
yeast  is  then  dried  in  the  sun  and  prepared  for  sale. 

The  rice  used  for  the  manufacture  of  the  yeast  is  not  of 
the  very  first  quality ;  grains  which  are  broken  may  even  be 
used. 


INDUSTRIAL   APPLICATION   OF  MALTASE.  235 

In  Cochin-China  the  manufacture  of  Chinese  yeast  is  car- 
ried on  everywhere  in  the  same  way.  In  Cambodia  and 
China  the  rice  is  sometimes  replaced  by  the  flour  of  beans  or 
maize. 

Native  Distilleries. — The  native  distilleries  do  not  de- 
mand a  complicated  outfit  any  more  than  do  the  yeast  manu- 
factories. The  plant  is  composed  of  a  shed  covered  with  a 
tiled  roof.  Under  this  roof  are  ranged  furnaces  in  parallel 
lines  separated  by  spaces  containing  basins  full  of  water,  in 
which  the  receivers  serving  to  condense  the  alcoholic  vapors 
are  plunged.  The  furnaces  measure  60  centimetres  in  height, 
1.2  m.  in  width  and  4  metres  in  length.  They  are  used  for  the 
heating  of  two  stills  and  a  boiler  devoted  to  cooking  the  rice. 
The  furnaces  are  heated  by  a  fire  of  mangle-wood. 

The  rice  used  for  making  the  mash  is  in  part  hulled  and 
mixed  with  a  certain  quantity  of  warm  water.  It  is  placed  in 
the  boilers,  which  are  covered  with  a  mat  and  a  sheet-iron 
cover.  In  each  boiler  are  placed  18  kilograms  of  grains  and 
22  kilograms  of  water,  and  they  are  cooked  for  2  hours.  The 
rice  is  at  this  time  completely  steeped.  It  is  then  spread  on 
mats  where  it  receives  the  Chinese  yeast  in  a  fine  powdery 
state,  after  which  it  is  placed  in  pots  of  about  20  litres  capac- 
ity, which  are  half  filled.  The  pots  are  closed  and  saccharifi- 
cation  allowed  to  occur.  When  the  starch  is  transformed, 
that  is,  at  the  end  of  about  3  days,  the  vessels  are  filled  with 
water;  fermentation  at  once  begins  and  at  the  end  of  48 
hours  the  action  is  finished.     The  contents  are  then  distilled. 

These  stills  are  formed  of  a  sheet-iron  vat,  a  wooden 
dome,  and  a  terra  cotta  head.     A  bamboo  tube,  2.5  m.  long 
and  inclined  at  450,  joins  the  still  to  the  condenser,  into  which 
it   conducts   the   alcoholic   vapors.     The   stills   are   placed 
directly  over  the  fire. 

The  residues  of  the  distillation  are  used  as  food  for  cattle. 

With  100  kilograms  of  rice  and  1.5  kilograms  of  Chinese 
yeast,  there  is  usually  obtained  60  litres  of  36  per  cent  al- 
cohol, or  18  litres  at  100  per  cent.     The  richness  of  the  first 


236  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

distillate  varies  according  to  the  distillery;  it  is  never  less 
than  340  or  over  420. 

Use  of  Moulds  in  Fermentation  Industries  in  Non- Asiatic 
Countries.  — From  the  point  of  view  of  the  utilization  of 
primary  materials,  Chinese  yeast,  as  well  as  Japanese  yeast, 
furnish  very  meagre  results  in  the  countries  of  their  origin. 

According  to  Atkinson,  the  yield  in  alcohol  in  the  manu- 
facture of  sake  reaches  only  50  to  56  per  cent  of  the 
theoretical  yield. 

Chinese  yeast  does  a  still  less  satisfactory  work.  Accord- 
ing to  Calmette,  100  kilograms  of  hulled  rice,  having  from  81 
to  84  per  cent  of  starch,  furnishes  about  18  litres  of  alcohoL 
in  the  distilleries  of  Cochin-China. 

This  unsatisfactory  result  must  be  attributed  in  great  part 
to  the  inefficiency  of  the  plants  as  well  as  to  slovenliness  in. 
the  labor. 

At  first  sight,  it  must  be  admitted  that  the  work  by  the 
aid  of  moulds  is  capable  of  being  improved  and  of  giving  in- 
dustrial results  similar,  and  perhaps  superior,  to  those  of  the 
ordinary  method. 

Moreover,  the  use  of  moulds  presents  great  advantages. 
The  work  appears  to  be  much  more  simple ;  the  yeast  and. 
the  malt  are  done  away  with  and  replaced  by  a  mould  which 
is  very  easily  cultivated  and  less  sensitive  than  malt  and  yeast 
to  the  action  of  heat  and  of  the  medium.  But  to  render 
practicable  the  use  of  moulds  it  is  first  necessary  to  give  up 
Oriental  methods,  adapt  oneself  to  the  conditions  of 
European  distilleries,  and  try  to  develop  a  practical  process. 

This  question  has  been  studied  by  the  Japanese  chemist 
Takamine,  and  also  by  Collette  and  Boidin. 

Takamine  has  been  engaged  in  the  application  of  moulds 
to  the  fermentation  industry  for  ten  years.  At  first  he  par- 
ticularly sought  a  medium  suitable  for  the  development  of 
Aspergillus  orizce,  which,  in  Japan,  is  cultivated  exclusively  on 
steamed  hulled  rice. 

To  furnish  the  mineral  element  to  the  plant,  a  certain: 


INDUSTRIAL   APPLICATION  OF  MALTASE.  237 

quantity  of  the  ash  of  Cornelia  Japonica  is  generally  added. 
Takamine  replaced  the  ash  by  an  addition  of  1  to  4  per  cent 
of  the  weight  of  the  grains  of  a  mixture  of  salts  in  which  are 
ammonium  tartrate  and  phosphate,  potassium  sulphate, 
and  magnesium  sulphate. 

According  to  the  author,  this  addition  of  salts  consider- 
ably increases  the  yield  and  has  the  further  advantage  of  per- 
mitting the  rice  to  be  replaced  by  other  cereals. 

To  prepare,  industrially,  cultures  of  Aspergillus  orizce, 
Takamine  proposes  the  following  process : 

Steam  the  grains  until  the  starch-cells  are  swollen,  cool, 
sprinkle  with  the  solution  of  salts;  mix  the  grains  well  and 
sow  with  Aspergillus  orizce.  The  cereals  thus  sown  are  left 
at  a  temperature  of  300  for  24  to  36  hours. 

The  lumps  formed  are  broken  up  and  the  grains  placed 
on  plates,  which  are  left  in  a  damp  atmosphere  until  the  com- 
plete maturity  of  the  moulds.  The  mouldy  mass  then  dried 
at  a  low  temperature  is  sifted.  Thus  the  spores  are  sepa- 
rated, which,  again  dried  at  a  moderate  temperature  and  then 
mixed  with  inactive  materials,  serve  as  agents  of  fermenta- 
tion. 

Takamine  also  makes  a  kind  of  malt  which  he  calls  talca- 
koji.  For  the  preparation  of  this  substance  he  prefers  to  use 
bran  or  brewery  or  distillery  malts,  and  proceeds  as  follows: 

The  raw  materials  are  sterilized  by  steam  and  sown  with 
the  spores  of  Aspergillus  orizce  at  a  temperature  of  300. 
One  gram  of  spores  is  used  for  50  kilograms  of  raw  material. 
The  development  of  the  mould  takes  place  in  a  very  damp 
malt-house  at  a  temperature  of  20°-30°.  After  24  hours' 
stay  in  the  malt-house,  the  mass  is  spread  in  thin  layers  and 
the  plant  is  allowed  to  grow.  Generally  its  development  is 
sufficient  after  4  or  5  days.  Then  the  material  is  dried  at  a 
temperature  not  exceeding  500. 

Takamine  also  recommends  for  taka-koji  to  separate  the 
spores  from  the  material  by  sifting  in  a  silk  sieve.  These 
spores  act,  according  to  him,  as  agents  of  alcoholic  fermenta- 


238  THE  ENZYMES  AND    THEIR   APPLICATIONS. 

tion,  while  the  taka-koji  itself  acts  as  agent  of  saccharifica- 
tion. 

Takamine  also  proposes,  with  much  reason,  to  use  for 
saccharification  a  clear  infusion  prepared  with  taka-koji.  For 
this  purpose  he  makes  a  cold  extraction  of  the  active  matter 
and  decants  the  liquid,  which  is  used  as  saccharifying  agent, 
while  the  solid  part  is  submitted  to  a  cooking  which  allows  of 
utilizing  the  starch. 

Taka-koji  serves  in  the  manufacture  of  a  ferment  useful 
in  the  distillery  and  the  bakery.  To  prepare  it,  bran  of  corn 
or  other  cereals  is  mixed  with  3  to  10  per  cent  of  taka-koji 
and  4  to  8  volumes  of  water.  The  mass  is  kept  at  65  °  for  15 
to  30  minutes  and  then  brought  to  a  boil.  Then  it  is  cooled 
to  6o°;  a  new  portion  (3  to  10  per  cent)  of  taka-koji  is  added 
and  a  second  saccharification  allowed  to  go  on.  This  ended, 
the  liquid  is  separated  from  the  solid  matter  by  nitration  or 
decantation,  the  mash  is  sterilized  and  sown  with  the  spores 
of  the  moulds.  A  fermentation  is  produced  which  lasts  from 
12  to  16  hours.  When  it  is  ended,  the  ferment  is  deposited 
at  the  bottom  of  the  vats  in  the  form  of  a  pasty  material 
which  is  pressed  and  used  in  various  industries. 

In  distillery  work,  according  to  Takamine's  system,  the 
following  is  the  manner  of  procedure: 

The  raw  materials,  grains,  potatoes,  etc.,  are  cooked  un- 
der pressure.  The  starch  is  then  saccharified  by  means  of 
taka-koji.  Saccharification  goes  on  for  an  hour  at  a  tem- 
perature of  65°-70°  and  the  quantity  of  taka-koji  used  is  from 
3  to  20  per  cent  of  the  quantity  of  grain  used,  according  to 
the  amount  of  diastase  the  koji  contains. 

Saccharification  accomplished,  the  mash  is  cooled  to  190 
and  leaven  is  added. 

For  the  preparation  of  the  leaven,  a  mash  of  cereals 
cooked  under  pressure  and  saccharified  by  taka-koji  is  used. 
The  saccharification  of  this  mash  is  accomplished  in  two 
stages.  First  it  is  saccharified  at  6o°  for  an  hour  and  then 
slowly  cooled  to  190.     A  new  portion  of  taka-koji  is  added, 


INDUSTRIAL   APPLICATION  OF  MALTASE.  239 

as  well  as  a  little  leaven  from  a  previous  operation,  and  it  is  al- 
lowed to  ferment. 

Greatly  attenuated  mashes  as  well  as  yeast-mother  are 
used  for  leaven  in  succeeding  operations. 

Generally  2  to  10  litres  of  leaven  are  used  for  100  litres  of 
mash  submitted  to  fermentation. 

Under  a  patent  taken  out  in  1894,  Takamine  proposes  to 
use  industrially  the  active  substances  of  moulds  by  precipitat- 
ing them  in  solid  state  from  their  solutions. 

To  cultivate  the  moulds,  malts,  bran,  or  other  amylaceous 
substances  are  used.  The  culture  made,  these  substances 
are  reduced  to  powder  and  soaked  in  cold  water  to  extract 
the  maltase.  The  liquid,  separated  from  insoluble  sub- 
stances, is  filtered  and  precipitated  with  1  to  3  volumes  of 
alcohol.  The  product  thus  obtained  is  placed  on  a  filter, 
washed  with  alcohol,  then  with  ether,  and  dried  at  a  moder- 
ate temperature.  According  to  Takamine,  the  active  sub- 
stance obtained  by  this  process  can  advantageously  replace 
malt  in  the  distillery  and  the  brewery. 

Takamine  also  advises  (and  this  he  holds  to  be  im- 
portant) the  addition  to  the  active  infusion,  before  the  ad- 
dition of  alcohol,  of  an  infusion  of  raw  materials,  as  bran, 
malts,  raw  grains,  etc.  According  to  him,  the  activity  of  the 
precipitated  enzymes  is  considerably  increased  by  this  opera- 
tion. 

From  the  description  of  the  process,  it  appears  attractive, 
and  we  have  hastened  to  repeat  the  experiment  of  Taka- 
mine; but  the  results  we  have  obtained  are  not  very  encour- 
aging. 

Aspergillus  orizee  contains  a  peptonizing  ferment  which 
acts  strongly  on  albuminoid  materials.  The  infusion  ob- 
tained is  very  viscid,  refuses  to  filter,  and  the  precipitate  re- 
sulting from  treatment  with  alcohol  does  not  show  very  ac- 
tive properties. 

The  addition  of  infusions  of  bran,  of  malts  or  of  raw  grains 
increases  the  saccharifying  power  of  the  diastases  of  Asper- 


240  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

gillus  oriscB,  as  we  demonstrated  before  Takamine,  but  it  does 
not  heighten  the  liquefying  power.  In  other  words,  the  in- 
crease is  apparent  rather  than  real.*  Under  a  more  recent 
patent,  Takamine  proposes  a  system  for  the  cultivation  of 
moulds  and  the  preparation  of  active  liquid  which  deserves 
mention. 

To  secure  a  great  surface  for  the  culture,  without  wasting 
the  nutritive  materials,  he  places  porous  objects  (fragments 
of  pumice-stone,  etc.)  in  a  nutritive  solution  and  allows  the 
moulds  to  develop  on  these  foundations.  This  very  ingen- 
ious idea  has  since  been  taken  up  by  Collette  and  Boidin.  To 
produce  industrially  a  growth  of  Amylomyces  Rouxii  straws 
are  saturated  with  the  nutritive  liquid,  sterilized,  and  then 
sown  with  the  mould.  To  favor  its  development  he  keeps  up 
a  strong  current  of  air  through  the  mass. 

By  this  means  and  with  relatively  little  nutritive  sub- 
stance, an  abundant  vegetation  is  obtained. 

The  fermenting  of  grain  by  Amylomyces  is  accomplished, 
according  to  the  method  of  Collette  and  Boidin,  in  the  follow- 
ing manner :  The  amylaceous  materials,  added  to  twice  their 
weight  of  water,  are  cooked  for  3  hours  under  a  pressure  of 
3!  to  4  atmospheres.  The  cooked  mass  is  placed  in  contact 
with  fresh  crushed  malt,  at  a  temperature  not  exceeding  700. 

The  weight  of  the  malt,  estimated  in  barley,  is  from  1^  to 
2  per  cent  of  the  total  weight  of  the  amylaceous  materials 
used  in  the  work.  Liquefaction  by  the  malt  continues  for 
about  an  hour.  The  mash  is  then  sterilized  in  a  great  diges- 
ter where  a  pressure  of  2  atmospheres  is  maintained,  after 
which  it  is  inoculated  and  allowed  to  ferment. 

This  fermentation  goes  on  in  special  vats  furnished  with 
agitators  and  injectors  of  air  and  steam.  The  boiling  mash, 
coming  from  the  sterilizer,  is  introduced  into  vats,  con- 
structed in  such  a  way  that  all  infection  can  be  avoided.  The 
cooling  of  the  mash  takes  place  in  the  fermentation  vats, 

*See  Comptes  Rendus  de  l'Academie  des  Sciences,  1892,  vol.  CIX, 
page  1324. 


INDUSTRIAL   APPLICATION   OF  MALTASE.  241 

where  it  falls  to  a  temperature  of  380  and  is  neutralized. 
Neutrality  of  medium  is  really  indispensable  to  the  normal 
development  of  Amylomyces.  The  vats  are  then  sown  with 
cultures  of  Amylomyces,  grown  on  a  small  quantity  of  amy- 
laceous materials,  after  which  sterilized  air  is  injected  and  the 
agitator  is  used. 

This  agitation  is  to  prevent  the  mould  from  developing 
on  the  surface,  because  if  it  grows  in  that  way  it  will  consume 
the  sugar  of  the  mash. 

After  20  hours,  the  development  of  the  mould  is  at  a 
maximum.  Then  it  is  cooled  to  38°-33°  and  sowed  a  second 
time  with  a  pure  yeast  culture.  This  yeast  produces  the  al- 
coholic fermentation.  The  mould,  to  yield  the  same  result, 
would  take  much  longer.  At  the  end  of  three  days  the  fer- 
mentation is  sufficiently  advanced,  and  the  mash  ready  for 
distillation. 

Critical  Examination  of  Oriental  Processes. — The  at- 
tempts made  by  Takamine,  Collette,  and  Boidin  to  introduce 
the  use  of  moulds  into  the  fermentation  industries  has  called 
forth  many  critical  articles  which  have  appeared  in  different 
reviews  of  distilling  and  brewing.  The  technical  journals 
seem,  on  the  whole,  very  reserved  as  to  the  value  of  the  new 
method.  In  general,  when  we  consider  an  industrial  pro- 
cess, the  only  criterion  which  we  can  admit  is  the  practical 
results  to  which  it  leads.  Now,  as  regards  the  use  of  moulds 
in  distilling,  results  of  this  kind  are  lacking,  at  present. 
With  the  exception  of  a  few  establishments  where  the 
inventors  have  been  experimenting  with  their  process, 
not  a  distillery  is  known  which  is  run  entirely  by  the  new 
method.  Under  these  circumstances,  it  would  be  premature 
to  pronounce  definitely  on  the  value  of  this  method  of  manu- 
facture. We  have  nevertheless  sought  to  compare  the  dif- 
ferent patents  of  Takamine  with  those  of  Collette  and  Boidin, 
since  they  have  been  considered  to  stand  for  different  pro- 
cesses. This  comparison  has  not  led  us  to  any  definite  con- 
clusions. 


242  THE  ENZYMES  AND    THEIR   APPLICATIONS. 

-Takamine's  method,  although  seven  years  earlier  than 
that  of  Collette  and  Boidin,  has  certain  points  of  resemblance 
to  the  latter  which  may  easily  lead  to  confusion  of  the  two. 

A  reading  of  the  patents  relative  to  the  use  of  moulds  re- 
veals in  a  striking  way  the  extravagant  hopes  of  the  inven- 
tors and  the  illusions  which  possessed  them  as  to  the  scope 
of  their  discovery. 

Takamine,  in  his  patents  of  1891,  claims,  as  his  exclusive 
right,  the  use  in  fermentation  industries  of  any  mould 
capable  of  producing  saccharification  and  fermentation  of 
amylaceous  materials  or  even  one  of  these  transformations 
alone. 

Seven  years  later,  Collette  and  Boidin  also  aspired  to 
this  exclusive  right.  They  claim,  as  the  result  of  their  re- 
searches, the  use  of  all  moulds  which  are  both  saccharifying- 
and  fermenting. 

One  might,  possibly,  condone  the  simplicity  of  the 
Japanese  chemist,  who  is  evidently  little  acquainted  with  our 
literature,  but  the  same  excuse  does  not  exist  for  the  French 
chemists.  The  use  of  moulds,  as  well  as  yeasts,  has  for  a 
long  time  been  public  property.  It  is  allowable  to  patent  a 
special  method  of  working  with  moulds,  but  not  the  principle 
of  their  use. 

In  reading  the  patent  it  is  very  hard  to  see  in  what  the 
invention  of  Collette  and  Boidin  really  consists.  One  might, 
perhaps,  characterize  their  process  by  the  sterilization  of  the 
mashes  and  the  development  in  them  of  pure  cultures. 

Takamine,  indeed,  sterilizes  only  the  leaven  and  then 
produces  fermentation  in  the  mashes  saccharified  at  high 
temperature.  But,  by  an  incomprehensible  misconception, 
Collette  and  Boidin  return,  in  additional  patents,  to  their 
method  of  work  and  claim  that  sterilization  of  the  mashes  can 
be  omitted.  So  this  is  not  the  distinctive  principle  of  their 
process. 

One  might  also  get  the  impression  by  studying  some  of 
the  patents  of  the  inventors,  that  Takamine  used  Aspergillus 


INDUSTRIAL   APPLICATION  OF  MALTASE.  243 

orizce  exclusively,  and  that  Collette  and  Boidin  used  only 
Amylomyces.  But  when  all  the  works  which  they  have  pub- 
lished are  reviewed,  this  is  found  not  to  be  so. 

It  is  to  be  regretted  that  Takamine  did  not  limit  his  pre- 
tensions to  Aspergillus  orizce,  which  would  have  rendered  his 
process  unquestionably  superior  to  that  of  his  competitors. 

On  the  whole,  the  practical  interest  of  moulds  centers  in 
their  saccharifying  powers. 

By  reducing  the  quantity  of  malt  for  the  preparation  of 
leaven,  it  has  been  possible  to  reduce  the  cost  of  the  yeast  so 
low  that  there  remains  little  or  nothing  to  be  done  in  this 
direction.  On  the  contrary,  an  economy  in  the  malt  used  for 
saccharification  of  the  mashes  would  afford  a  real  advantage. 

Aspergillus  orizce  is  unquestionably  a  more  active  pro- 
ducer of  diastase  than  Amylomyces  Rouxii,  and  from  this  point 
of  view  it  is  of  much  more  interest  to  distillers.  Japanese 
yeast  affords  still  other  advantages  over  Amylomyces  Rouxii. 
Aspergillus  orizce  secretes  not  only  maltase  but  also  sucrase. 
It  can  consequently  be  used  in  molasses  and  beet-sugar  dis- 
tilleries where  Amylomyces  Rouxii  would  be  of  no  use. 

The  process  of  Collette  and  Boidin  does  not  yield  mashes 
containing  more  than  4  to  5  per  cent  of  alcohol,  while  Asper- 
gillus orizce  produces  alcohol  up  to  12  per  cent  or  more. 

Furthermore,  with  Aspergillus  orizce  there  is  no  need  of 
a  special  equipment,  while  the  Amylomyces  Rouxii  system 
cannot  be  adopted  without  a  complete  and  costly  refitting 
of  the  plant. 

These  faults  are  peculiar  to  the  Collette  and  Boidin  pro- 
cess, but  there  are  others  common  to  the  two  methods. 

1.  Moulds  are  oxidizing  agents  and,  as  such,  always 
cause  great  losses  of  carbohydrates. 

2.  Alcohol  produced  with  moulds  has  a  peculiar  taste  and 
contains  many  more  impurities  than  that  resulting  from  the 
use  of  good  yeasts. 

3.  Moulds  generally  furnish  very  limited  quantities  of  dia- 
stase, and  to  obtain  a  satisfactory  result  an  abundant  culture 


244  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

must  be  allowed  to  develop  in  the  mashes,  which  necessarily 
influences  the  yield  of  alcohol. 

One  may  conclude  from  the  preceding  observations  first 
of  all  that  the  activity  of  workers  is  not  at  all  restrained 
by  patents  taken,  and,  further,  that  great  improvements 
must  be  introduced  in  processes  using  moulds  for  them  to 
become  practicable.  It  will  be  necessary  to  study  thor- 
oughly the  conditions  of  development  of  the  moulds  in  ques- 
tion and  it  will  be  necessary,  also,  by  a  systematic  acclima- 
tization, to  make  them  produce  a  diastatic  secretion  which 
shall  be  more  active  and  less  sensitive  to  the  conditions  of 
the  medium. 

Up  to  the  present  time  it  has  been  the  capitalists  in  par- 
ticular who  have  been  occupied  with  the  question;  it  is  to  be 
hoped  that  disinterested  investigators  will  apply  themselves 
to  it  in  their  turn. 

BIBLIOGRAPHY. 

Atkinson. — Sur  la  diastase  du  koji.     Monit.  scientifique,  1882. 

The  Chemistry  of  Sake  Brewing  in  Japan,  Tokio,  1881;  Nature,  1878; 

Chemical  News,  April,  1880. 
Eijkmann. — Mikrobiologisches    tiber    die    Arrakfabrikation    in    Batavia. 

Centralblatt  fur  Bakt.  und  Paras.,   1894. 
Went  und  Geerligs. — Uber  Zucker  und  Alcoholbildung  durch  Organis- 

men    bei    der    Verarbeitung    der    Nebenprodukte    der    Rohrzucker- 

fabrikation.     Wochensch.  fur  Brauerei,  1894. 
Hofmann. — Mittheilungen    der    deutschen    Gesellschaft    fur    Natur-    und 
M.  O.  Korschelt. — Memoires  de  la  Societe  asiatique.     Berlin,  1878.     See 

Volkerkunde  Ostasiens.     Heft,  6. 

Dinglers  Polytech.  Journal,  1878. 
Ahlburg. — Mittheilungen  der  deutsch.     Gesellschaft  fur  Natur-  und  Vol- 
kerkunde Ostasiens.     December,   1878. 
Ikula. — Sakefabrikation.      Chemik.   Zeitung,   1890. 
Kellner. — Chemik.  Zeitung,  1895. 
A.    Calmette. — La    fabrication    des    alcools    de    riz    en    Extreme-Orient, 

Saigon,  Imprimerie  coloniale,  1892. 
Mori    Nagaoka. — Beitrag    zur    Kentniss    der    invertirenden    Fermente. 

Zeit.  fur  physiol.  Chemie,  1890. 
Juhler.— Centralbl.  fur  Bakter.,  1895. 
Jorgensen—  Centralbl.  fur  Bakter.,  1895. 
Wehmer.— Centralbl.  fur  Bakter.,  1895. 


INDUSTRIAL  APPLICATION  OF  MALTASE.  245 

Klocker    und   Schionning.— Centralbl.    fur   Bakter.,    1895. 

Dr.  Liebscher.— Ueber  die  Benutzung  des  Gahrungspilzes  Eurot. 
orizae.     Zeitschrift  fur  Spiritus  Indust.,  1881. 

Kosai  Tabe. — Centralbl.  fur  Bakter.,  n,  p.  619. 

Bodin  et  Rolants.— Contribution  a  l'etude  de  l'utilisation  de  l'Amylomyces 
Rouxii.     La  biere  et  les  boissons  fermentees,  1897. 

Petit— Quelques  procedes  nouveaux  en  Distillerie.  Moniteur  scien- 
tifique,   1898. 

Sorel—  Comptes  rendus  de  deux  congres  de  chimie  appliquee.  Paris 
1897. 

Comptes  Rendus,  1895. 

Nititenski. — Moisissures  saccharificant  l'amidon.  Technitscheski  sbornick. 
La  biere  et  les  boissons  ferm.,  1898. 

Takamine.— Brevet  No.  216840,  19  octobre  1891.  Perfect,  dans  la  produc- 
tion des  ferments  alcooliques. 

Brevet  No.  214033,  3  av.  1891. 

Brevet   No.    241322,    11    sept.    1894.     Conversion   des    matieres   amy- 

lacees  en  sucre. 

Brevet    No.   241321,    11    sept.    1894.     Perf.    dans    la    preparation    des 

mouts  fermentes. 

Brevet  No.  241323,  11  sept.     Fabric,  du  Tako  Koji. 

Collette  et  Boidin. — Brevets  Nos.  258084,  265245,  130172  en  1896.  Pro- 
cede  d'utilisation  des  moisissures  pour  l'extraction  des  residus  de 
l'alcool.  France,  15  juillet  1896,  No.  125722,  certif.  d'additus.  11 
janv.  1897. 


CHAPTER  XX. 

ENZYMES  ACTING  ON  CARBOHYDRATES. 
TREHALASE. 

Trehalase  is  an  enzyme  which  acts  on  trehalose,  an 
isomeric  sugar  corresponding  to  the  formula 

C12H2201X  +  2H0O. 

This  sugar  plays  the  part  in  plants  of  a  reserve  substance.. 
Wigers  and  Mitscherlich  have  found  it  in  spurred  rye  and 
Berthelot  in  the  Trehala  of  Syria.  It  is  frequently  found 
in  great  quantity  in  fresh  fungi,  from  whence  it  almost  en- 
tirely -disappears  during  drying.  For  example,  it  consti- 
tutes 10  per  cent  of  the  dry  matter  of  Agaricus  muscarius. 
Trehalose  does  not  reduce  Fehling's  solution,  and  is  trans- 
formed into  glucose  by  the  action  of  acids.  A  similar  hydra- 
tion may  be  obtained  by  the  use  of  an  enzyme,  trehalase, 
discovered  by  Bourquelot. 

This  worker  discovered  the  presence  of  the  enzyme  in 
■Aspergillus  niger  and  Penicillium  glaucum,  as  well  as  in  other 
fungi.  This  enzyme  is  also  found  in  malt,  and  in  the  small 
intestine. 

The  transformation  of  trehalose  into  glucose  may  be 
expressed  by  the  following  equation: 

C12H2201]L  +  H20  =  2C6H1206. 


The  diastatic  action  may  be  followed  by  the  change  of 
rotatory  and  reducing  powers  of  the  liquid. 

Trfehalose  has  a  rotatory  power  of  (00*198°,  while  the 
rotatory  power  of  glucose  is  only  (a)d  52.40. 

246 


ENZYMES  ACTING   ON   CARBOHYDRATES.  247 

Experiments  with  trehalase  may  be  made  in  a  2  per  cent 
solution  of  trehalose,  at  a  temperature  of  33°-35°. 

Trehalase  is  much  more  sensitive  to  the  action  of 
heat  than  maltase.  At  540  its  action  is  checked  and  at  640 
the  enzyme  is  completely  destroyed.  The  reactions  of  the 
medium  also  have  a  very  great  influence  on  trehalose.  An 
acidity  corresponding  to  2  to  4  milligrams  of  sulphuric  acid 
seems  to  favor  the  transformation  of  trehalose  bv  the  en- 
zyme, but  if  the  amount  of  acid  is  increased,  the  activity  dim- 
ishes,  and  with  0.2  grams  the  action  of  the  enzyme  is  almost 
stopped. 

According  to  Fischer,  an  infusion  of  malt  may  produce 
the  decomposition  of  trehalose,  while  the  salivary  diastase, 
ptyalin,  has  not  this  property. 

Amylase,  precipitated  and  purified  according  to  the 
method  of  Lintner,  acts  energetically  on  trehalose.  By  leav- 
ing, at  a  temperature  of  350,  10  cubic  centimetres  of  a  10  per 
cent  solution  of  trehalose  with  a  half-gram  of  amylase,  the 
formation  of  0.5  grams  of  glucose  has  been  observed. 

Emil  Fischer  recognized  trehalase  in  Frohberg  yeast. 
This  enzyme  is  retained  in  the  cells  of  this  yeast  and  with 
difficulty  passes  into  the  surrounding  medium.  For  this 
reason  an  aqueous  extract  of  yeast  does  not  possess  the 
property  of  transforming  trehalose,  while  in  the  cells  of  yeast 
trehalose  is  transformed  into  glucose. 

By  adding  5  grams  of  yeast  to  1  gram  of  trehalose  dis- 
solved in  10  cubic  centimetres  of  water,  Fischer  was  able  to 
find,  after  40  hours  action  at  a  temperature  of  330,  the  form- 
ation of  0.2  grams  of  reducing  sugar. 

According  to  this  author,  the  existence  of  trehalase  is  to 
be  doubted  and  he  believes  that  it  is  amylase  which  produces 
the  transformation  of  trehalose  into  glucose. 

According  to  Fischer,  therefore,  amylase  must  have  the 
property  of  acting  on  starch,  giving  maltose,  and  on  an 
isomer  of  maltose,  giving  glucose. 


248  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

To  prove  the  existence  of  trehalase  we  have  made  the 
following  experiment: 

Equal  quantities  of  yeast,  cultivated  in  sterilized  wort, 
are  added,  under  like  conditions,  to  a  solution  of  dextrins 
and  to  a  solution  of  trehalose.  The  2  solutions  are  left  for 
2  days  at  300  and  then  tested.  For  these  experiments  2 
grams  of  yeast  are  used,  25  cubic  centimetres  of  a  1  per  cent 
solution  of  soluble  starch  and  20  cubic  centimetres  of  a  10 
per  cent  solution  of  trehalase.  The  action  of  the  yeast  takes 
place  in  the  presence  of  chloroform. 

The  solution  of  trehalase  yields,  under  these  conditions^ 
0.34  grams  of  glucose,  while  in  the  solution  of  soluble  starch 
no  traces  of  sugar  are  found.  The  enzyme  secreted  by  the 
yeast  is  therefore  not  amylase,  and  the  fact  that  the  diastase 
of  Lintner  acts  on  trehalose  proves  only  that  this  diastase 
contains  other  enzymes  than  amylase. 

LACTASE. 

Pasteur  has  demonstrated  that  sugar  of  milk  treated  with 
mineral  acids  is  transformed  into  galactose  and  dextrose  ac- 
cording to  the  equation: 

C12H22O11  +  H20  =  C6H1206  +  C6H1206. 

Lactose.  Dextrose.  Galactose. 

In  living  cells,  the  transformation  of  lactose  is  carried  on 
by  means  of  an  enzyme  which  causes  the  same  action  as  the 
acid. 

The  existence  of  this  ferment  was  doubted  for  a  long 
time  and  the  transformation  of  the  sugar  of  milk  in  the  or- 
ganism attributed  to  vital  activity.  Beijerinck  was  the  first 
to  discover  the  presence  of  lactase  in  certain  yeasts  found  in 
cheese  and  kephir.  Since  then  Duclaux,  De  Kayser,  and 
Adametz  have  found  other  species  of  yeasts  which  secrete 
the  same  diastase.  Emil  Fischer,  repeating  the  experiment 
of  Beijerinck,  has  confirmed  the  fact  that  the  filtered  in- 
fusion of  kephir  acts  upon  lactose. 


ENZYMES  ACTING   ON  CARBOHYDRATES.  249 

As  in  kephir  the  Saccharomyces  act  in  symbiosis  with 
other  micro-organisms,  it  was  of  interest  to  find  out  whether 
the  enzyme  is  secreted  by  the  yeast  or  by  the  accompanying- 
bacteria.  The  experiments  which  Fischer  made  with  this 
object  have  established  the  following  facts: 

1  st.  Certain  alcoholic  yeasts  are  capable  of  fermenting 
lactose;  2nd.  The  action  of  a  yeast  on  milk-sugar  depends 
solely  on  its  power  to  secrete  lactase. 

The  enzyme  acting  on  the  lactose  is  retained  inside  the 
cells  and  passes  with  great  difficulty  into  the  surrounding 
medium.  Even  when  the  cells  of  yeast  are  crushed  with 
powdered  glass,  it  is  difficult  to  extract  the  active  substance. 
The  diffusion  of  the  diastase  of  the  cells  is  accelerated  by 
chloroform.  Lactase  can  be  precipitated  from  its  solutions 
by  alcohol  without  completely  losing  its  activity. 

The  action  of  lactase  on  lactose  can  be  determined  by  the 
aid  of  the  polariscope ;  by  the  transformation  of  the  lactose 
into  dextrose  and  galactose  the  rotation  of  the  solution  in- 
creases about  a  third. 

Lactose  and  dextrose  have  a  rotatory  power  of  {a)d  -f- 
52.5,  while  that  of  galactose  is  (a)d  +  83. 

INULASE. 

Certain  plants  contain,  as  reserve  substance,  a  carbo- 
hydrate called  inulin. 

These  plants  generally  contain  at  the  same  time  an  ac- 
tive principle  which  transforms  this  carbohydrate  into  an  as- 
similable sugar. 

This  enzyme  was  discovered  by  J.  R.  Green,  who  named 
it  '•  inulase." 

The  presence  of  inulase  has  been  observed  in  the  tubers 
of  Jerusalem  artichokes  during  their  development,  in  Asper- 
gillus niger,  in Penicillium  glaucum,  and  in  the  tubers  of  dahlias. 
According  to  Bourquelot,  it  is  supposed  that  this  enzyme  is 
also  found  in  chicory,  garlic,  and  onions,  as  well  as  in 
many  other  vegetables. 


250  THE  ENZYMES  AND    THEIR   APPLICATIONS. 

By  the  action  of  inulase,  inulin  is  hydrated  and  trans- 
iormed  into  levulose  according  to  the  formula: 

(C6H10O5)18  +  18H0O  =  i8C6H1206. 

Inulin.  Levulose. 

According  to  Green,  this  transformation  is  accomplished 
•  by  a  progressive  hydration  of  the  inulin,  with  formation  of 
intermediate  substances.  Granting  that  the  molecule  of 
inulin  is  very  complex,  one  may  assume  that,  during  hydra- 
tion, there  are  formed,  besides  levulose,  different  inulins  hav- 
ing different  molecular  weights. 

However,  inulin,  after  having  undergone  a  partial  hydra- 
tion, possesses  the  same  rotatory  power  as  before  under- 
going the  action  of  the  enzyme. 

The  existence  of  intermediate  substances  is  somewhat 
problematical,  as  the  formation  of  these  bodies  by  the  action 
of  acids  has  not  been  determined.  The  optimum  tempera- 
ture of  inulase  is  found  between  500  and  6o°.  The  action  of 
the  enzyme  is  influenced  by  the  reaction  of  the  medium. 
In  a  neutral  liquid,  or  with  0.005  Part  °f  hydrochloric  acid, 
hydration  progresses  regularly.  In  the  presence  of  increas- 
ing amounts  of  acid,  the  activity  of  the  enzyme  decreases. 
With  0.2  of  acid  or  1.5  of  sodium  carbonate,  the  diastase 
is  destroyed. 

The  influence  of  the  reaction  of  the  medium  is  more 
strongly  shown  at  400  than  at  io°-i5°. 

The  transformation  of  inulin  into  levulose  may  be  fol- 
lowed either  by  observation  of  the  rotatory  power  or  by  that 
of  the  reducing  power. 

Inulin  has  a  rotatory  power  of  (a)d  —  36,  while  levulose 
gives  a  rotation  to  the  left  which  is  almost  double. 

In  the  distilleries  which  use  Jerusalem  artichokes  as  a 
raw  material,  the  inulin  has  to  be  inverted  when  a  satisfac- 
tory yield  in  alcohol  is  desired.  To  effect  this  transforma- 
tion, the  use  of  barley  malt  is  advised. 


ENZYMES  ACTING   ON  CARBOHYDRATES.  251 

This  method  is  very  poor  indeed,  for  amylase  is  without 
action  on  inulin  and  malt  does  not  contain  inulase. 

The  transformation  of  inulin  into  levulose  can  be  brought 
about  very  easily:  it  is  sufficient  to  cook  the  raw  materials 
under  low  pressure  to  effect  a  complete  inversion. 

PECTASE. 

In  the  pulp  of  carrots  and  beets,  and  also  in  the  soft 
parts  of  fruits,  Fremy  found  a  reserve  substance  to  which 
he  gave  the  name  of  pectose. 

This  substance  is  insoluble  in  water  and  in  alcohol.  It 
very  much  resembles  cellulose. 

Pectose  undergoes  a  succession  of  transformations  dur- 
ing the  ripening  of  fruits:  it  is  transformed  into  pectin  and 
finally  into  pectates. 

Pectin  is  a  neutral  substance  which  gives  with  water 
a  viscous  solution  from  which  it  is  precipitable  by  alcohol. 

The  transformation  of  pectose  into  pectin  is  very  prob- 
ably produced  by  an  enzyme  which,  however,  has  not  yet 
been  isolated. 

The  transformation  of  pectin  into  pectates  is  better 
known,  and  the  intervention  of  an  enzyme  is  here  definitely 
established. 

This  active  substance  is  called  pectase.  This  name  ought 
to  belong  to  the  substance  acting  on  pectose  and  not  to  the 
enzyme  transforming  pectin.  This  latter  enzyme,  according 
to  the  correct  nomenclature,  should  rather  be  called  "  pec- 
tinase." 

The  composition  of  pectin  is  not  definitely  established. 
According  to  Fremy  it  has. the  formula: 

and  according  to  Chandnew: 

The  mechanism  of  the  reaction  produced  by  the  pectase 
is  little  known. 


25 2  THE  ENZYMES  AND   THEIR  APPLICATIONS. 

It  is  not  even  clearly  established  that  the  reaction  is 
caused  by  hydration,  and  it  may  well  be  that  the  mechanism 
of  the  reaction  consists  in  a  molecular  change  of  the  same 
nature  as  that  found  in  the  transformation  of  sugar  into 
lactic  acid. 

The  action  of  pectase  on  a  solution  of  pectin  is  shown 
by  the  gelatinization  of  the  solution,  and  by  the  formation  of 
a  reducing  substance. 

Bertrand  and  Mallevre  have  shown  that  the  reaction 
takes  place  only  in  the  presence  of  certain  salts.  A  solution 
of  pure  pectin,  with  the  addition  of  pectase,  free  from  cal- 
cium salts,  never  becomes  gelatinous. 

The  solidification  of  the  solution  takes  place  instantly  if 
to  the  mixture  are  added  several  drops  of  a  solution  of  cal- 
cium chloride,  a  substance,  which,  without  pectase,  could  not 
produce  gelatinization. 

The  calcium  salt  may  be  replaced  by  salts  of  barium  or 
strontium,  which  play  just  the  same  part. 

To  obtain  a  solution  of  pectase,  carrots  gathered  in  the 
process  of  growth  are  used,  because  it  is  then  that  these 
plants  contain  the  most  diastase.  It  is  well  to  pare  the 
carrots  and  use  only  the  central  part,  as  the  skin  contains, 
but  little  pectase. 

The  substance  is  reduced  to  a  pulp  and  the  juice  ex- 
tracted by  pressure;  in  this  way  70  to  80  per  cent  of  a  turbid 
liquid  is  obtained,  which  is  filtered  after  addition  of  a  little 
chloroform. 

This  liquid  is  very  active  in  a  solution  of  pure  pectin.  To 
preserve  the  filtered  solution  of  pectase,  precipitate  the  salts 
of  lime  and  magnesium  by  the  addition  of  alkaline  oxalate, 
previously  determining  by  analyses  the  amount  of  oxalate  to 
use.  The  quantity  of  salts  contained  in  the  juice  varies 
slightly  with  the  species  of  carrots;  for  three  different 
samples  Bertrand  obtained  the  following  figures: 

Lime 0.016%         0.018%         0.013% 

Magnesia 0.029  0.021 


ENZYMES  ACTING   ON   CARBOHYDRATES.  253 

It  is  well,  in  practice,  to  use  a  third  of  alkaline  oxalate 
in  excess  of  the  amount  calculated  for  the  salts  present. 
The  solution  of  pectase  to  which  oxalate  has  been  added 
rapidly  becomes  clear  and,  after  filtration,  gives  a  trans- 
parent solution. 

This  product  can  be  kept  for  a  long  time  if  chloroform  is 
added  and  it  is  put  fresh  into  full  bottles  sheltered  from  the 
light.  Jelly  is  not  produced  in  a  solution  of  pectin  free  from 
salts. 

To  prepare  pectase  in  a  pure  state,  clover  is  used.  The 
plants  are  crushed  in  an  iron  mortar;  the  mass  is  then 
pressed,  and  the  juice,  with  chloroform  added,  is  placed  away 
from  the  light.  At  the  end  of  24  hours,  a  coagulum  is  pro- 
duced in  the  liquid  which  permits  of  filtration.  In  the  filtered 
liquid  the  diastase  is  precipitated  by  alcohol  as  has  been 
done  with  the  other  enzymes. 

Measurement  of  Pectase.— Pectase  is  very  widely  dis- 
tributed in  the  vegetable  kingdom.  It  is  found  in  stems, 
flowers,  and  leaves  of  different  plants. 

Bertrand  and  Mallevre  propose  the  following  method  for 
measuring  it: 

To  one  volume  of  a  2  per  cent  pectin  solution  add  one  vol- 
ume of  the  juice  under  examination  and  measure  the  dia- 
static  power  by  the  time  required  for  the  liquid  to  become 
gelatinized. 

The  results  obtained  with  the  juices  of  different  plants 

are  given  below: 

Tomatoes 48  hours. 

Grape-vine 24 

Carrots 2 

Maize  (leaves) 8 

Qover 10  minutes. 

By  using  this  method,  Bertrand  and  Mallevre  studied 
the  influence  of  the  medium  on  pectase. 

Different  samples  of  the  same  solution  of  pectin  were 


254  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

acidified  in  different  degrees  and  the  same  quantity  of  pec- 
tase was  added: 

Hydrochloric  Acid.  Coagulation 

Per  cent.  at  the  end  of 

o        |     hour. 

0.02 I 

0.06 3!  hours. 

0.1     20       " 

Pectase  is  unfavorably  influenced  by  the  acid  'reaction 
of  the  medium ;  0.06  per  cent  of  acid  in  the  liquid  produces 
a  delay  of  three  hours  in  coagulation.  However,  acid  does 
not  easily  destroy  this  active  substance. 

By  neutralizing  the  acid  solutions,  which  have  become 
weak  and  slightly  active,  new  liquids  are  obtained  which  act 
very  rapidly.  This  resistance  of  pectase  to  the  acid  reaction 
explains  why  the  action  of  pectase  is  weak  in  green  fruits: 
before  ripening  the  enzyme  is  in  the  presence  of  a  large 
amount  of  acid  and  does  not  act,  or  acts  only  very  feebly, 
while  during  ripening  the  acidity  disappears  and  the  action 
of  the  pectase  is  shown  with  much  more  intensity. 

CYTASE. 

Cellulose  is  often  assimilated  by  vegetable  cells.  This 
assimilation  is  preceded  by  liquefaction  and  a  more  or  less 
complete  transformation.  The  agent  which  produces  the 
change  is  cytase. 

As  celluloses  exist  whose  properties  differ  considerably, 
it  is  necessary,  at  the  outset,  to  assume  also  the  existence  of 
different  cellulose-dissolving  enzymes. 

Sachs  was  the  first  to  discover  that,  during  the  germina- 
tion of  the  stones  of  dates,  the  cellulose  of  the  endosperm 
is  gradually  dissolved  and  that  the  products  formed  are  ab- 
sorbed by,  the  young  plants  which,  with  the  cellulose,  pro- 
duce the  transitory  starch. 

Green,  by  treating  germinated  date-seeds  with  glycerin, 


ENZYMES  ACTING   ON   CARBOHYDRATES.  255 

obtained  an  active  solution  which  causes  swelling  as  well 
as  partial  solution  of  certain  celluloses. 

The  destruction  of  vegetable  tissues  by  moulds  must  also 
be  attributed  to  a  secretion  of  cytases;  however,  the  isolation 
of  these  enzymes  is  accomplished  with  much  difficulty  and 
their  existence  was  doubted  for  a  long  time. 

The  difficulty  met  with,  when  it  is  desired  to  isolate  this 
diastase,  comes  probably  from  its  decomposition.  It  is 
probable  that  these  enzymes  are  destroyed  as  rapidly  as 
they  appear,  and  that  it  is  for  this  reason  that  they  are  not 
found  accumulated  in  the  cells. 

A  *more  stable  cytase  was  discovered  by  Brown  and 
Morris  in  malt  dried  in  the  air.  To  obtain  this  enzyme  in  a 
solid  state,  an  infusion  of  malt  is  precipitated  with  alcohol, 
and  the  precipitate  dried  in  vacuo. 

The  product  obtained  contains,  besides  amylase,  a  cellu- 
lose-dissolving enzyme. 

The  activity  of  this  ferment  is  shown  by  its  property  of 
dissolving  the  cellulose  envelope  of  grains  of  starch.  This 
may  be  verified  by  causing  it  to  act  on  the  endosperm  of 
barley.  For  this,  very  thin  shavings  of  barley  endosperm 
are  put  into  an  infusion  of'  malt  and  it  is  found  that  the  cel- 
lular walls  soften  and  then  enter  partially  into  solution. 

Cytase  is  present  from  the  beginning  of  germination  of 
cereals,  appearing  even  before  amylase. 

The  dissolving  action  of  cytase  during  malting  is  exer- 
cised upon  the  whole  of  the  endosperm  and,  as  a  result,  the 
germinated  grain  becomes  friable  and  mealy. 

This  transformation  may  also  be  produced  artificially  by 
placing  a  grain  of  barley  from  which  the  embryo  has  been 
removed  in  a  malt  infusion.  By  a  prolonged  stay,  the  en- 
dosperm changes  its  appearance  completely;  it  becomes 
mealy  and  friable,  while  if  the  infusion,  i-s  previously  heated 
to  6o°,  the  same  effect  is  not  obtained.  At  this  temperature, 
the  amylase  of  the  solution  does  not  lose  its  power  to  hydro- 
lyze  starch,  while  the  cytase  is  destroyed. 


256  THE  ENZYMES  AND   THEIR  APPLICATIONS. 

The  transformation  which  cytase  produces  during  germ- 
ination has  been  studied  from  a  chemical  point  of  view.  It 
is  very  probable  that  the  cellulose  is  transformed  into  sugar, 
but  it  may  also  be  that  the  action  of  the  cytase  is  less  com- 
plete. During  germination,  according  to  J.  Gruss,  the  cel- 
lular walls  are  only  partially  liquefied,  and  the  action  of 
cytase  is  reduced  to  freeing  the  amylaceous  cells  and  in- 
directly facilitating  the  action  of  amylase. 

By  cultivating  the  germ  of  cereals  in  different  media, 
Brown  and  Morris  found  that  the  presence  of  an  assimilable 
hydrocarbon  influences  unfavorably  the  secretion  of  cytase. 
They  also  found  that  a  slight  acidity  of  the  medium  is,  on  the 
contrary,  very  favorable  to  the  secretion. 

In  general,  all  the  conditions  which  favor  the  secretion 
of  amylase  are  equally  favorable  to  that  of  cytase. 

CAROUBINASE. 

Caroubinase  is  an  enzyme  acting  on  a  carbohydrate 
isolated  from  grains  of  the  Ceratonia  siliqua  to  which  we  have 
given  the  name  of  caroubin. 

This  enzyme  causes  a  liquefying  and  saccharifying  action 
on  the  endosperm  of  carob-seeds  and  plays  a  very  important 
part  during  the  first  period  of  the  development  of  this  plant. 

The  endosperm  of  the  seeds  of  Ceratonia  siliqua  is  found 
to  be  partially  composed  of  a  carbohydrate  which  occurs  in 
the  form  of  a  homogeneous  and  horny  mass,  not  colored  by 
iodine,  and  possessing  some  properties  like  those  of  agar- 
agar.  To  prepare  this  carbohydrate  in  a  pure  state,  the 
seeds  are  freed  of  their  exterior  envelope  as  well  as  their 
embryo,  and  the  endosperm  is  dissolved  in  warm  water.  The 
solution  is  then  precipitated  by  alcohol. 

The  operation  is  carried  out  in  the  following  manner: 
The  seeds  are  allowed  to  soak  for  five  or  six  days,  the  liquid 
being  renewed  three  or  four  times  a  day.  The  grains  swell  a 
great  deal  and  absorb  three  times  their  weight  of  water.     In 


ENZYMES  ACTING    ON   CARBOHYDRATES.  257 

this  state  it  is  easy  to  separate  the  endosperm  from  the  testa 
and  the  embryo.  One  hundred  grams  of  dry  seeds  furnish 
53  grams  of  albumen.  The  swelling  of  the  grain  during 
soaking  is  due  almost  entirely  to  the  mucilaginous  substance 
which  they  contain  and  which  constitutes  an  elastic  and  re- 
sisting mass. 

By  submitting  the  endosperm  to  the  action  of  warm  water, 
in  a  water-bath,  a  transparent  jelly  is  obtained  which  can  be 
filtered  through  a  silk  filter.  It  is  well  to  use  enough  water 
to  obtain  a  thick  syrup.  To  precipitate  the  caroubin,  add  to 
the  cooled  syrup  twice  its  volume  of  98  per  cent  alcohol. 
The  carbohydrate  is  thrown  down  in  long  filaments  which 
are  collected  on  a  cloth. 

The  first  precipitate  thus  obtained  contains  2  to  3  per 
cent  of  albuminoid  materials  and  salts  which  are  easily  elim- 
inated by  redissolving  the  product  in  water  and  again  precip- 
itating it  with  alcohol.  By  treating  the  endosperm  eight  to 
ten  times  successively  with  warm  water,  there  is  obtained  an 
almost  complete  extraction  of  the  carbohydrate  it  contains. 

The  product,  purified  and  dried  at  ioo°,  is  a  white  sub- 
stance, which  is  spongy,  very  friable,  and  having  the  chemical 
formula  of  celluloses.  Instead  of  alcohol,  one  may  just  as 
well  use  barium  hydrate,  which  precipitates  the  carbohydrate 
in  a  pure  state.  Caroubin  is  easily  hydrated  by  acids  as  well 
as  by  a  special  diastase,  caroubinase. 

To  isolate  this  enzyme,  we  used  an  infusion  of  germinated 
carob-beans.  A  hundred  grams  of  germinated  seeds  reduced 
to  a  paste  were  put  to  soak  in  water,  at  a  temperature  of  300, 
for  twelve  hours.  To  the  filtered  liquid  3  volumes  of  alcohol 
were  added ;  the  precipitate  was  washed  with  alcohol,  then 
with  ether,  then  dried  in  vacuo. 

The  active  substance  obtained  by  this  method  dissolves 
easily  in  water  and  gives  a  reaction  with  guaiacum  and  hydro- 
gen peroxide. 

Caroubinase  acts  energetically  at  400,  and  its  activity  in- 
creases with  the  temperature  up  to  500,  which  is  its  optimum 


258  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

temperature;  at  700,  the  action  becomes  very  weak  and  at 
8o°  the  enzyme  is  destroyed. 

Caroubinase  acts  very  slightly  in  a  neutral  medium.  An 
addition  of  0.01  to  0.03  per  cent  by  volume  of  formic  acid 
favors  the  action  of  the  enzyme. 

To  determine  the  diastatic  power  of  caroubinase,  the  de- 
gree of  fluidity  produced  in  a  jelly  of  caroubin  is  taken  as  a 
starting  point. 

The  diastatic  power  may  also  be  estimated  by  the  greater 
or  less  facility  with  which  the  liquid  may  be  filtered. 

The  solution  of  caroubin  not  transformed  by  the  enzymes 
does  not  pass  through  the  filter,  while  the  solution  of  carou- 
bin with  a  sufficient  quantity  of  diastase  added  passes 
through  very  rapidly. 

The  process  employed  is  as  follows : 

Pour  into  test-tubes  50  cubic  centimetres  of  water;  add 
0.1  c.c.  of  normal  formic  acid  and  1  gram  of  pulverized  carou- 
bin. Mix  and  add  to  different  tubes  2,  5,  7,  10,  15  cubic  cen- 
timetres of  the  liquid  to  be  examined.  If  there  is  room, 
bring  the  volume  up  to  65  cubic  centimetres  and  leave  it  for 
three  hours  at  45  °. 

All  the  samples  receive  the  same  amount  of  chloroform, 
and  the  experiments  are  conducted  in  duplicate :  on  the  one 
hand  with  fresh  infusion,  on  the  other  with  this  same  infu- 
sion previously  kept  for  a  half-hour  at  900. 

The  tubes  which  have  not  received  any  infusion,  or  in 
which  the  substance  has  been  destroyed  by  heating,  may  be 
turned  over  without  the  liquid  running  out,  while  the  tubes 
which  have  received  a  sufficient  quantity  of  enzyme  contain  a 
very  fluid  substance  which  easily  passes  through  the  filter. 

To  study  the  secretion  of  caroubinase,  we  allowed  the 
embryos  of  Ceratonia  siliqua  to  grow  in  varied  conditions  and 
followed  the  transformation  of  the  nutritive  materials  as  well 
as  the  quantity  of  diastase  formed. 

The  embryo,  separated  from  the  endosperm  and  culti- 
vated in  the  dark,  develops  very  slowly  and  gives,  after  eight 


ENZYMES  ACTING   ON  CARBOHYDRATES.  259 

to  ten  days,  a  rootlet  of  the  same  length  as  itself.  Then 
placed  in  calcareous  earth  and  in  the  light,  the  germ  develops 
into  a  puny  plant  which  generally  dies  at  the  end  of  three  to 
four  weeks. 

The  progress  of  growth  is  quite  different  when  the 
isolated  embryo  is  cultivated  in  hydrated  caroubin ;  the  ger- 
mination is  more  rapid ;  a  rootlet  of  the  length  of  the  seed  is 
obtained,  and  the  embryo,  set  out  in  the  earth,  rapidly 
develops  into  a  plant  of  several  branches. 

During  germination  away  from  the  light,  the  caroubin 
used  swells  a  great  deal  and  is  partly  liquefied,  but  the  quan- 
tity of  carbohydrate  absorbed  is  inconsiderable. 

The  liquefaction  and  absorption  of  the  caroubin  pro- 
gresses much  more  rapidly  as  soon  as  chlorophyll  appears  in 
the  plantlet.  The  embryo,  developed  in  the  dark  and  trans- 
planted in  a  calcareous  soil,  absorbs  in  three  or  four  days  a 
quantity  of  caroubin  equal  to  its  own  weight. 

By  taking  specimens  at  different  stages  of  germination, 
we  found  that  the  active  substance  appears  abundantly  when 
the  plantlets  are  completely  developed  and  that  the  enzyme 
becomes  more  active  when  the  chlorophyll  begins  to  appear. 

Caroubinase  is  both  a  liquefying  and  a  saccharifying 
agent.  When  jelly  of  caroubin  is  analyzed  immediately  after 
liquefaction,  the  liquid  is  found  to  contain  no  trace  of  reduc- 
ing-sugar.  Caroubin  liquefied  by  the  enzyme  is  easily  pre- 
cipitated by  alcohol,  but  the  precipitate  no  longer  has  the 
properties  of  the  caroubin.  It  is  strongly  dextro-rotatory 
and  easily  dissolves  in  water. 

By  a  prolonged  action  of  caroubinase  on  caroubin,  a  so- 
lution is  obtained  in  which  alcohol  no  longer  produces  pre- 
cipitate and  a  reducing-sugar  easily  fermenting  under  the  in- 
fluence of  beer-yeast  is  produced. 

BIBLIOGRAPHY. 

E.  Bourquelot. — Sur  un  ferment  soluble  nouveau  dedoublant  le  trehalose 
en  glucose.     Comptes  Rendus,  1893. 


26o  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

E.  Bourquelot. — Remarques  sur  le  ferment  soluble  seer,  par  l'aspergillus 

et  le  penicillium.     Soc.  biol.,  1893,  juin. 

Digestion  du  trehalose.    Soc.  biol.,  1895. 

Transformation  du  trehalose  en  glucose.    Bui.  de  la  Soc.  chim.  de 

Paris,   1893,  p.    192. 
Emil    Fischer. — Spaltung   von   Trehalose.     Berichte    der    deutsch.  chem. 

Gesellschaft,  1895,  p.  1433. 
Einfluss  der  Configuration  auf  die  Wirkung  der  Enzyme.     Berichte 

der  deutsch.  chemisch.  Gesellschaft,  1895,  2,  p.  1429. 
Beijerinck. — Centralbl.    fur    Bakt.    und    Parasitenkunde.       Zweite    Ab- 

theilung,   1898. 
E.  Bourquelot. — Inulase  et  fermentation  alcoolique  indirecte  de  l'inuline. 

Soc.  biol.,  Paris,  1893. 
G.    Dulle. — Ueber  die   Einwirkung  von   Oxalsaure   auf   Inulin.      Chem. 

Zeit.,  1895 
J.  R.  Green. — Annals  of  Botany,  1888,  1893. 
Bourquelot  and  H.  Herissey. — Sur  la  matiere  gelatineuse  (pectine)  de  la 

racine  de  gentiane.     Journ.  de  chim.  et  de  pharm.,  1898,  p.  473. 
Sur  l'existence,  dans  l'orge  germee,  d'un  ferment  soluble  agissant 

sur  la  pectine.      Comptes  Rendus,  1898,  p.  191. 
Fremy. — Memoire  sur  la  maturation  des  fruits.     Ann.  de  chim.  et  phys., 

1848,  XXIV,  p.  5. 
Schreibler. — Berichte  der  deut.  chem.  Gesellschaft,   B.  I.  p.  59 
Chandnew. — Liebigs  Annalen,  LI,  p.  355. 
Frid   Reintzer. — Ueber   die   wahre    Natur   der   Gumifermente.     Zeit.    fiir 

phys.  Chemie,  1890,  XIV. 
Wiesner. — Ueber    das    Gumiferment:    ein    neues    diastasisches    Ferment. 

Berichte,  1885,  p.  619. 
Cross. — Bull,  de  Soc.  chim.  de  Paris,  1896. 
Bertrand  and  Mallevre. — Recherches  sur  la  pectase  et  sur  la  fermentation 

pectique.     Bull,  de  la  Soc.  chim.  de  Paris,  1895,  XIII,  pp.  77,  252. 
Nouvelles  recherches  sur  la  pectase  et  sur  la  fermentation  pectique. 

Comptes  Rendus,  1895,  ier  semestre,  p.  no. 
Sur  la  diffusion  de  la  pectase  dans  le  regne  vegetal  et  sur  la  prepara- 
tion de  cette  diastase.     Comptes  Rendus,  1895,  CXXI,  p.  727. 
Reintzer. — Sur  la  diastase  qui  dissout  les  enveloppes  cellulosiques.     Zeit. 

fiir  physiol.  Chem.,  XXIII,  p.  175,  1897. 
Tromp.  de  Haas  and  B.  Tollens. — Recherches  sur  les  matieres  pectiques. 

Bull,  de  la  Soc.  chim.  de  Paris,  1895,  p.  1246. 
Brown  and  Morris. — Untersuchung  iiber  der   Keimung  einiger  Graser. 

Zeit.  fur  das  gesammte  Brauwesen,  1890. 
De      Bary. — Ueber     einige     Sclerotinen     und      Sclerotinenkrankheiten. 

Bot.  Zeit.,  1886. 
Schmulewitsche. — Ueber  das  Verhalten  der  Verdauungstoffe  zu  Rohfasser 

der    Nahrungsmittel.     Bull.    Acad,    des    sciences    Saint    Petersburg, 

t.  XL 


ENZYMES  ACTING   ON  CARBOHYDRATES.  261 

*Srtrt!:\z nouve' hydra,e  de  carbone' ia  caroubine-  c°m*- 

Sur  la  caroubinase.    Comptes  Rendus,  IX,  p.  764. 


CHAPTER  XXI. 

FERMENTS  OF  GLYCERIDES  AND  GLUCOSIDES. 

Saponifying  ferments. — Ferments  of  glycerides. — Serolipase  and  pancrea- 
tolipase. — Measurement  of  lipase. — Influence  of  temperature  and 
alkalinity  of  the  medium. — Differences  between  lipases  of  different 
origins. — Ferments  of  glucosides. — Myrosin,  Emulsin,  Rhamnase, 
Erythrozyme,  Betulase. 

FERMENTS  OF  GLYCERIDES. LIPASE. 

The  pancreatic  juice  has  the  property  of  splitting  fats  into 
fatty  acids  and  glycerin.  This  property  is  due  to  the  pres- 
ence of  a  soluble  ferment  to  which  has  been  given  the  name 
of  steapsin  or  lipase.  The  reaction  which  steapsin  causes 
may  be  represented  by  the  following  equation : 

C3H5(C18H3502)3  +  3H20  =  C3H5(OH)3  +  3C18H86Oa. 


Stearin.  Glycerin.  Stearic  acid. 

To  obtain  steapsin  in  solution,  the  pancreas  is  macerated 
in  a  solution  of  sodium  or  potassium  carbonate.  It  can  then 
be  extracted  from  the  pancreas  by  glycerin. 

The  pancreatic  juice  acts  upon  fats  as  a  saponifying  and 
emulsifying  agent.  The  emulsion  is  produced  by  the  pan- 
creatic juice,  owing  to  the  alkaline  reaction  and  to  the  vis- 
cosity of  the  liquid,  and  not  by  the  action  of  the  enzyme  con- 
tained therein. 

The  pancreatic  juice  as  well  as  the  products  of  maceration 
of  the  pancreas  contain  relatively  little  of  the  enzyme  and  the 
saponification  of  the  fatty  substances  is  always  incomplete. 

The  enzyme  of  the  pancreatic  juice  also  acts  upon  other 
substances  than  the  fats ;  it  attacks  the  lecithins,  decompos- 

262 


FERMENTS   OF  GLYCERIDES  AND   GLU  COS  IDES.  263- 

ing  them  into  glycero-phosphoric  acid,  choline,  glycerin,  and 
free  fatty  acids.  Steapsin  acts  also  on  some  other  ethers :  on 
the  benzoic  ether  of  glycerin,  and  on  phenyl  succinate,  as 
well  as  on  salol. 

It  decomposes  this  latter  body  into  salicylic  acid  and 
phenol. 

The  ferment  of  the  glycerides  is  very  abundant  in  the 
vegetable  kingdom.  Its  presence  has  been  observed  in  the 
poppy,  hemp,  maize,  rape-seeds,  as  well  as  many  other  plants. 

To  obtain  an  active  liquid  containing  lipase,  Green 
ground  the  germinated  seeds  of  the  castor-oil  plant  in  a  5  per 
cent  solution  of  sodium  chloride,  with  the  addition  of  a  small 
quantity  of  potassium  cyanide.  He  then  dialysed  the  liquid 
to  separate  the  salts  from  it.  This  solution,  mixed  with  an, 
emulsion  of  castor-oil,  soon  began  to  decompose  the  oil,  set- 
ting free  the  fatty  acid. 

An  active  substance  showing  all  the  properties  of  lipase 
is  met  with  in  Peiiicillium  glaucum. 

The  presence  of  a  similar  substance  called  seroiipase  is 
also  found  in  the  blood.  It  plays  an  important  part  in  the 
assimilation  of  fats.  Hanriot,  who  studied  this  enzyme  with 
much  care,  pointed  out  a  method  for  measuring  the  active 
substance  and  determined  the  influence  of  temperature,  and 
of  the  reaction  of  the  medium  upon  this  enzyme.  Accord- 
ing to  him  there  is  a  difference  between  the  lipase  of  the  pan- 
creatic juice  and  the  lipase  of  the  blood. 

Measurement  of  Lipase. — To  measure  the  lipase,  Hanriot 
and  Camus  make  use  of  a  solution  of  monobutyrin. 

They  take  1  cubic  centimetre  of  the  liquid  containing  the 
lipase  to  be  measured,  add  to  it  10  cubic  centimetres  of  a  1 
per  cent  solution  of  monobutyrin.  The  solution  is  carefully 
neutralized  with  sodium  carbonate,  then  heated  to  250  for  20 
minutes.  Under  the  influence  of  lipase,  the  liquid  becomes 
acid,  and  this  acidity  is  estimated  by  again  neutralizing  the 
solution  with  sodium  carbonate:  the  number  of  drops  used 
serves  to  measure  the  diastatic  activity. 


264  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

The  solution  of  sodium  carbonate  used  for  the  saturation 

is  prepared  in  such  a  way  that  each  drop  of  the  alkaline  liquid 

neutralizes  0.000001  of  a  gram-molecule  of  acid.     The  dia- 

static  power  is  expressed  by  the  number  of  millionths  of  a 

gram-molecule  of  acid  freed  during  20  minutes  at  250;  1 

cubic  centimetre  of  serum,  for  example,  is  said  to  possess  a 

diastatic  force  of  33  if,  in  20  minutes  at  25 °,  it  frees  a  quan- 

33  X88 

tity  of  butyric  acid,  molecular  weight  88,  equal  to - 

1,000,000 

Influence  of  Temperature  and  the  Reaction  of  the  Me- 
dium.— Heat  exerts  a  considerable  influence  on  the  activity 
of  lipase.  Between  o°  and  500  it  acts  with  an  increasing  en- 
ergy, but  beyond  that  point  the  diastatic  activity  begins  to 
diminish  and  the  enzyme  is  soon  destroyed. 

Temperature  Quantities  saponified, 

of  the  reaction.  In  10  minutes.  In  I  hour. 

o° 4-5   J3-5 

10 

20 6.7  29.3 

25 io-i    35- 

37 13-5   39-5 

40 16.9  56.5 

50 22.6  71.2 

60 27.1    36.1 

70 22.6 22.6 

The  temperature  of  6o°  appears  favorable  at  the  begin- 
ning, but  in  the  end  destroys  the  diastase. 

The  influence  of  temperature  on  lipase  may  be  shown  by 
warming  the  serum  to  different  temperatures  and  causing  it 
to  act  then  on  monobutyrin  at  370. 

Serum  heated.  Diastatic  activity. 

5o°-55° 4i-5 

6o°-62° , 0.7 

65°-66° Extremely  slight. 

7o°-72° '...  o 


FERMENTS  OF  GLYCERIDES  AND   GLUCOS1DES. 


265 


The  action  of  lipase  is  proportional  to  the  quantity  of 
enzyme  used,  at  least  at  the  beginning  of  the  action.  The 
following  table  shows  this  fact : 


Duration  of  the  Action. 

Quantity  of  Lipase. 

0.5  c.c. 

6 

12.5 

20 

30 

I  c.c. 

1.5  c.c. 

2  c.c. 

20  minutes 
1  hour. 

1  h.  20  m. 

2  hours 

II 

25 

36 

54 

16 

37 
53 
73 

22 

48 
62 

66 

Cessation  of  the  proportionality  is  noticed,  in  the  case  of 
lipase  as  well  as  in  that  of  other  diastases,  when  the  action 
is  prolonged  or  takes  place  at  high  temperatures. 

The  glycerin  and  the  sodium  butyrate  formed  during  the 
action  have  no  influence  at  all  on  the  diastatic  activity;  the 
presence  of  monobutyrin  is  also  almost  without  effect  on  the 
saponification. 

The  alkalinity  of  the  medium  influences  considerably  the 
course  of  saponification  by  serolipase. 

Hanriot,  to  show  this  action,  made  the  following  ex- 
periment: 

To  indentical  mixtures  of  serum,  monobutyrin,  and  water 
(10  cubic  centimetres)  were  added  varying  amounts  of 
sodium  carbonate.  After  twenty  minutes  he  determined  the 
quantity  of  monobutyrin  saponified  by  neutralizing  with 
sodium  carbonate.     He  obtained  the  following  results: 


Excess  of  carbonate  of  sodium  in  milli- 
grams        o       2 

Activity  of  the  lipase 22     33 


4       6       8     10     15     2» 
40     44     46     52     74     86 


Difference  between  Lipases  of  Different  Origin. — Hanriot, 
having  remarked  that  the  ablation  of  the  pancreas  in  the  or- 
ganism does  not  prevent  the  secretion  of  lipase,  attributed  to 
the  blood  the  property  of  secreting  a  lipase  different  from 
that  of  the  pancreas.  He  called  it  serolipase,  in  distinction 
from  the  first,  which  he  called  pancreatolipase. 


266  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

But  the  ablation  of  the  pancreas  is  a  very  delicate  opera- 
tion and  impossible  to  perform  without  leaving  active  frag- 
ments of  the  gland.  Lipase,  on  the  other  hand,  may  be  pre- 
served in  the  blood.  The  existence  of  two  lipases  needs, 
therefore,  to  be  clearly  established. 

Hanriot  sought  to  differentiate  the  two  enzymes  by  their 
mode  of  action  and  their  sensitiveness  to  physical  and  chem- 
ical agents.  For  that,  he  prepared  two  solutions  having  the 
same  activity,  that  is,  producing  the  same  quantity  of  butyric 
acid  by  acting  on  monobutyrin  during  the  same  time. 

These  two  solutions  should,  therefore,  if  there  exists 
merely  a  single  lipase,  contain  the  same  quantity.  Now, 
when  the  action  of  the  serolipase  and  pancreatolipase  is  pro- 
longed for  20  minutes,  it  is  observed  that  the  serum  enzyme 
produces  a  quantity  of  butyric  acid  double  that  obtained  by 
the  pancreatolipase. 

On  the  other  hand,  the  enzyme  of  the  pancreas  acts  with 
great  difficulty  in  an  acid  medium,  while  serolipase  produces 
a  very  energetic  transformation  under  the  same  conditions. 

Pancreatic  juice.  Serum. 
Activity  in  alkaline  medium  (excess  of  sodium  carbonate, 

per  litre  0.2  gr.) 23  22 

Activity  in  acid  medium 9  16 

The  serolipase  and  the  pancreatolipase  act  differently  at 
the  same  temperatures:  two  solutions  of  these  enzymes  pos- 
sessing the  same  activity  at  140  gave  the  following  figures  at 
other  temperatures : 

c-       ,.  Pancrea- 

Serohpase.      ,   ,. 

c  tohpase. 

At  15° ti  10 

"30° '• 15  10 

"  42  ° 21  11 

It  is  seen  by  this  table  that  the  action  of  pancreatolipase 
is,  up  to  a  certain  limit,  independent  of  temperature,  while 
serolipase  produces  a  much  more  energetic  action  at  420 
than  at  150. 


FERMENTS   OF  GLYCERIDES   AND   GLUCOSIDES.  267 

Finally,  the  two  enzymes' differ  as  regards  stability.  In 
fact,  serolipase  remains  unchanged  during  whole  months, 
while  the  pancreas  enzyme  becomes  inactive  at  the  end  of 
a  few  days. 

The  lipases  of  the  pancreas  and  the  serum  act  differently, 
therefore,  at  the  same  temperatures  and  are  influenced  dif- 
ferently by  the  reaction  of  the  medium.  Furthermore,  they 
present  different  characteristics  of  stability.  These  proper- 
ties, however,  are  not  enough  to  demonstrate  that  seroli- 
pase and  pancreatolipase  are  two  very  distinct  chemical 
substances.  In  the  case  of  lipase,  as  we  have  seen  for  amylase 
and  glucase,  the  conditions  of  the  medium  produce  varia- 
tions in  the  properties  of  the  diastase.  The  foreign  sub- 
stances found  in  the  blood,  as  well  as  the  extractive  materials 
of  the  pancreas,  give  different  characteristics  to  the  two  dia- 
static  extracts,  but  the  enzyme  is  really  the  same  in  the  two 
cases. 

FERMENTS  OF  GLUCOSIDES. 

Glucosides  are  combinations  of  sugars  and  organic  sub- 
stances containing  one  or  more  hydroxyls. 

Glucosides  exist  in  which  the  sugar  is  found  combined 
with  alcohols,  phenols,  aldehydes  or  organic  acids.  These 
ethers  are  frequently  found  in  plants,  especially  in  the  bark 
and  roots. 

The  manner  of  formation  of  glucosides  in  living  cells  is  as 
yet  little  known.  It  is  very  probable  that  their  formation  is 
due  to  a  molecular  concentration  followed  by  a  dehydration 
which  is  produced  by  special  enzymes. 

According  to  Gautier,  the  formation  of  certain  glucosides 
may  be  explained  by  a  transformation  of  formic  aldehyde : 

i2CH20  +  H2  =  C12H1(i07  +  5H20. 

Formic  aldehyde.  Arbutin. 

i3CH20  +  2H0  =  C,,H1807  +  6H20. 

Formic  aldehyde.  Salicin. 


268  THE  ENZYMES  AND   THEIR  APPLICATIONS. 

The  part  played  by  glucosides  in  the  cells  is  also  little 
known  at  the  present  time. 

In  some  cases  they  evidently  #play  the  part  of  reserve 
materials.  In  other  cases  the  assimilation  of  the  products  of 
cleavage  of  glucosides  appears  of  little  probability.  In  fact, 
these  bodies  contain,  besides  sugar,  poisonous  substances 
which  must  act  unfavorably  on  the  cells. 

In  the  parts  of  plants  where  the  presence  of  glucosides  is 
observed,  there  are  almost  always  found  enzymes  under  the 
influence  of  which  these  ethers  are  hydrated,  then  split,  re- 
generating the  sugar.  The  enzymes  of  glucosides  are  gen- 
erally enclosed  in  special  cells  which  separate  them  from  the 
substances  on  which  they  can  act. 

The  glucoside-splitting  enzymes  have  this  peculiarity  that 
they  act,  not  on  a  single  body,  as  is  the  case  with  sucrase,  for 
example,  but  on  a  whole  series  of  bodies. 

Their  action  may  be  exerted  on  numerous  ethers  result- 
ing from  the  combination  of  glucose  with  bodies  belonging" 
either  to  the  fatty  series  or  the  aromatic  series. 

Emulsin. — By  treating  bitter  almonds,  reduced  to 
powder,  with  water,  an  aromatic  oil  is  produced  which  did 
not  exist  in  the  almonds  before  the  treatment. 

This  reaction  is  caused  by  an  enzyme,  emulsin,  on  a 
special  substance  contained  in  the  almond :  amygdalin.  The 
reaction  may  be  represented  by  the  following  equation: 

GMH,TNOn  +  2H20  =  2C6H1206  +  C7HeO  +  HCN. 

Amygdalin.  Glucose.  Benzoic  aldehyde.  Hydrocyanic 

acid. 

Emulsin  and  also  amygdalin  were  discovered  by  Robiquet 
and  Boutron. 

This  diastase  is  found  in  the  leaves  of  cherry-laurel  as  well 
as  in  sweet  almonds.  With  the  latter,  oil  of  bitter  almonds 
is  not  obtained  on  account  of  the  absence  of  amygdalin. 

Bourquelot  discovered  the  presence  of  emulsin  in  fungi. 
Fungi  parasitic  on  trees,  especially,  contain  great  quantities 


FERMENTS  OF  GLYCERIDES  AND    GLUCOSIDES.  269 

of  this  substance;  thus  he  discovered  the  presence  of  this 
enzyme  in  Poly  poms  sulfurcus,  in  Armillaria  mellea,  and  in 
Polypoms  fomcntarhis. 

Emulsin  has  also  been  met  with  in  Penicillinm  glaucum,  in 
Aspergillus  niger,  as  well  as  in  other  moulds. 

Emulsin  acts  on  a  great  number  of  glucosides,  causing 
the  reactions  expressed  by  the  following  equations : 

C12H1607  +  H20  =  C6H1206  -f  C6H602. 

Arbutin.  Glucose.  Hydroquinone. 

With  helicin,  a  product  of  oxidation  of  salicin : 
Ci3Hi607  -f-  H20  =  C6H12Oe  +  C7H602. 

Helicin.  Glucose.         Salicylic  aldehyde. 

With  salicin,  extracted  from  the  bark  of  poplar  or  the 
flowers  of  Spirea  ulmaria : 

Ci3H1807  +  H20  =  C6H1206  +  C7H802. 

Salicin.  Glucose.  Saligenin. 

With  phloridzin,  extracted  from  the  bark  of  the  apple- 
tree  : 

C21H24O10  +  H20  =  C6H12Oe  +  C15H1403. 

Phloridrin.  Glucose.  Phloretin. 

With  daphnin,  extracted  from  the  Daphne  gnidium : 
C15H1609  +  H20  =  C6H1206  +  C9H604. 

Daphnin.  Glucose.  Daphnetin. 

With  coniferin,  extracted  from  the  Larix  Europe?: 
C1CH^208  +  H20  =  C6H1206  +  C10H12O3. 

Coniferin.  Glucose.  Coniferic  alcohol. 

With  esculin  of  2Esculus  hippocastannm,  which  certain 
authors  consider  as  an  isomer  of  daphnin,  glucose  and  esculi- 
tin  are  obtained : 

QoHicOj,  -f-  H20  =  C6H12Og  +  C9H604. 

Esculin.  Glucose.  Esculitin. 


2  7°  THE  ENZYMES  AND   THEIR  APPLICATIONS. 

Emulsin  acts  also  on  the  chlorinated  and  brominated  de- 
rivatives of  the  glucosides. 

According  to  Fischer  emulsin  can  also  transform  lactose 
into  galactose  and  dextrose.  But  this  assertion  needs  to  be 
verified,  for  it  is  very  probable  that  the  emulsin  having  served 
for  these  experiments  contained  a  certain  proportion  of  lac- 
tase. 

Emulsin,  which  acts  on  bodies  very  differently  from  a 
chemical  point  of  view,  acts  differently  on  the  various  mono- 
saccharids,  according  to  their  configuration.  Thus,  while  it 
acts  on  the  /3-methyldextro-glucoside,  it  is  without  action  on 
the  <*-methyldextro-glucoside. 

In  living  plants,  amygdalin  is  not  transformed  because  it 
is  localized  in  special  cells,  and  is  thus  separated  from  the 
glucosides.  Mechanical  action  is  necessary  to  bring  the  two 
bodies  into  contact. 

Thus  the  transformation  of  amygdalin  into  oil  of  bitter 
almonds  and  hydrocyanic  acid  occurs  very  rapidly  when  the 
plants  containing  the  glucoside  and  the  enzyme  are  macer- 
ated in  water. 

According  to  Guignard,  the  emulsin  cells  are  located  in 
the  cotyledons.  In  the  cherry-laurel  the  enzyme  is  local- 
ized in  the  cells  of  the  endodermis. 

Emulsin  gives  characteristic  reactions  with  the  solution 
•of  orcin  as  well  as  with  Millon's  reagent.  With  the  latter, 
the  vegetable  cells  containing  emulsin  are  colored  an  orange- 
red.  When  the  cells  containing  emulsin  are  carefully  heated 
with  a  solution  of  orcin,  a  violet  coloration  is  obtained.  This 
solution  is  prepared  by  adding  2  cubic  centimetres  of  hydro- 
chloric acid  to  a  1  :  10  solution  of  orcin. 

The  physical  and  chemical  conditions  of  the  action  of 
emulsin  are  but  little  known. 

Chloral,  up  to  3.5  per  cent,  does  not  influence  the  course 
of  hydration  by  emulsin,  but  the  enzyme  is  sensitive  to  the 
action  of  8  per  cent  alcohol. 

Neutral  salts  do  not  appear  to  influence  the  course  of  the 


FERMENTS   OF  GLYCE RIDES  AND    GLUCOSIDES.  271 

hydration.  Alkaline  salts,  on  the  contrary,  have  a  retarding 
influence. 

Emulsin  plays  an  important  part  in  the  manufacture  of 
the  oil  of  bitter  almonds  as  well  as  in  the  manufacture  of 
laurel-water. 

To  manufacture  the  oil  of  bitter  almonds,  the  almonds 
are  reduced  to  powder,  the  oil  extracted,  water  added,  and 
the  mass  left  at  the  ordinary  temperature  for  the  reaction  to 
take  place.  Fermentation  ended,  they  are  distilled  with 
steam. 

To  obtain  a  good  yield  one  must  avoid  beginning  to  distill 
before  the  fermentation  is  finished. 

For  the  manufacture  of  laurel-water,  the  fresh  leaves  of 
the  plant  are  used.  They  are  crushed,  cold  water  is  added, 
and  finally  distilled. 

It  is  necessary  to  leave  cold  water  for  some  time  in  con- 
tact with  the  leaves  before  heating. 

Emulsin  is  used  in  pharmacy,  where  it  is  prepared  in  the 
following  manner : 

Sweet  almonds  are  blanched,  powdered,  and  submitted  to 
a  strong  pressure  which  presses  out  the  oils.  The  press- 
cakes  are  put  to  soak  in  three  times  their  volume  of  water; 
the  mass  is  again  pressed  and  thus  is  obtained  an  oily  liquid 
which  is  clarified  by  leaving  it  for  some  time  at  a  temperature 
of  300. 

Then  the  upper  layer  of  the  liquid,  which  consists  of  oil,  is 
removed  and  the  enzyme  in  the  clear  liquid  is  precipitated  by 
alcohol.  The  precipitate  is  collected  on  a  filter,  washed  in 
95  per  cent  alcohol,  and  dried  in  vacuo. 

Thus  a  yellowish  powder  is  obtained,  very  rich  in  phos- 
phates and  mineral  salts.  Completely  dried,  it  can  be  heated 
to  ioo°  without  losing  its  activity. 

Emulsin  is  soluble  in  water  and  in  a  dry  state  remains  un- 
changed for  a  long  time. 


272  THE    ENZYMES  AND    THEIR  APPLICATIONS. 


MYROSIN. 

Myrosin  was  discovered  by  Bussy  in  mustard-seed. 

The  characteristic  odor  of  black  mustard-seed,  when 
ground  with  water,  is  due  to  the  presence  and  action  of  this 
enzyme. 

Myrosin  is  very  widely  distributed  in  the  vegetable  king- 
dom ;  it  is  frequently  found  in  plants  of  the  family  Cmcifercc. 
This  diastase,  like  emulsin,  is  located  in  special  cells  scattered 
in  the  different  organs  of  the  plant,  but  chiefly  in  the  root  and 
the  leaves. 

It  acts  on  sinigrin  or  potassium  myronate,  which  is  de- 
composed by  hydration.  This  chemical  reaction  is  generally 
considered  as  taking  place  according  to  the  equation : 

C10H18NKS2O10  =  C6H1206  +  C3H5NSC  +  KHS04. 

Sinigrin.  Glucose.'        Allyl  iso-thiocyanate.  Potassium 

bisulpbate. 

According  to  this  equation,  the  decomposition  would  be 
produced  without  hydration.  But  free  myronic  acid  has  not 
yet  been  studied;  it  is  very  probable  that  potassium  myro- 
nate has  the  formula: 

C10H16KNS2O9  +  H20, 

and  it  is,  therefore,  probable  that  the  diastase  produces  a 
hydration  and  not  a  simple  decomposition. 

In  the  seeds  of  white  mustard  myrosin  is  also  found,  but 
the  sinigrin  is  replaced  by  another  glucoside,  sinalbin.  The 
reaction  produced  may  be  expressed  by  the  following  equa- 
tion: 

C30H44N2S2016  = 

Sinalbin. 

C6H1206  +  C7H70-NCS  +  C16H24N05  —  HS04. 

Glucose.  Oxy-benzyl-thiocyanate.  Sinapine  sulphate. 


FERMENTS  OF  GLYCE  RIDES  AND   GLU  COS  IDES.  273 

Myrosin  may  also  act  upon  many  other  glucosides.  To 
this  diastase  is  attributed  the  formation  of  essential  oils  of 
different  plants  such  as:  water-cress,  Reseda  odorata,  and 
Cochlioria  officinalis. 

RHAMNASE. 

This  enzyme  is  found  in  the  fruits  of  the  Avignon  berry 
(Rhamnus  infectoria).  It  acts  on  a  yellow  coloring  matter 
having  the  characteristics  of  a  glucoside,  xanthorhamnin, 
and  transforms  it  into  rhamnetin  and  isodulcite : 

C24H32014  +  3H20  =  C12H10O3  +  2C6H14Oe. 

Xanthorhamnin.  Rhamnetin.  Isodulcite. 

ERYTHROZYME. 

This  diastase  is  secreted  by  the  root  of  the  madder.  It 
acts  on  a  glucoside  of  alizarin :  rubian,  which  is  also  found  in 
fresh  madder-root.  The  reaction  probably  occurs  according 
to  the  following  equation : 

C26H28014  +2H20  -  2C6H1206  +  C14H804. 

Rubian.  Glucose.  Alizarin. 


BETULASE. 

Betulase  is  met  with  in  the  bark  of  Betula  lenta.  This 
enzyme  acts  on  gaultherin  and  the  reaction  may  be  expressed 
by  the  following  equation: 

Ci4Hi808  +  H20  =  C0H12O6  +  C6H4/   q    oqw 

Gaultherin.  Glucose.  Methyl  salicylate. 

To  prepare  this  enzyme,  take  the  bark  of  the  Betula  lenta 
and  reduce  it  to  powder,  treat  it  with  4  volumes  of-glycerin 
and    leave   it   at    the    ordinary   temperature    for   30   days. 


274  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

The  mass  is  then  pressed,  and  the  enzyme  precipitated 
from  the  solution  with  5  volumes  of  alcohol.  The  deposit  is 
filtered  off,  washed,  and  dried. 

A  kilogram  of  bark  gives  with  this  treatment  nearly  a 
gram  of  enzyme. 

Betulase  does  not  color  with  tincture  of  guaiacum  and 
does  not  act  on  other  glucosides  than  gaultherin. 


BIBLIOGRAPHY. 

CI.  Bernard. — Legons  de  physiologie  experimentale. 

Dobelle. — Actions  du  pancreas  sur  les  grains  et  l'amidon.     Proceed,  of 

the  Royal  Soc,  t.  XIV. 
Duclaux. — Diastase  du  pancreas.     Microbiolog.  Encyclop.  Chim.,  1883,  p. 

153- 
Sur  la  digestion  des  matieres  grasses   et  des   celluloses.     Comptes 

Rendus,   1882. 
Sigmundi. — Ueber  die  Fettspaltenden  Fermente  in  Pflanzen.     Akad.  der 

Wissen.  Wien,   1890-1891. 
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p.  753- 
Sur  la  repartition  de  la  lipase  dans  l'organisme.     Comptes  Rendus, 

1896,  p.  833 

■ Sur  la  dosage  de  la  lipase.     Comptes  Rendus,  1897,  p.  235. 

Sur  la  non-identite  des  lipases  d'origines  differentes.     Comptes  Ren- 
dus, 1897,  p.  778. 
Gerard.— Sur    une    lipase    vegetale    extraite    du    penicillium    glaucum. 

Comptes  Rendus,  1897,  p.  37°. 
Robiquet. — Journal  de  pharmacie,  2  mai,  1838. 
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■ Journal  de  pharmacie,  XXIV,  p.  326. 

Bussy. — Note    sur   la    fermentation    de    l'huile    essentielle    de    moutarde. 

Comptes  Rendus,  1839,  p.  815. 
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Berichte  der  deut.  chem.     Gesellschaft,  1895,  2031. 


FERMENTS  OF  GLYCE RIDES  AND   CLUCOSIDES.  275 

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deut.  chem.     Gesellschaft,  1894,  p.  864. 
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chim.,  1896,  6e  seri,  t.  Ill,  p.  117. 
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So.  nat.  Boton,  1887,  p.  118. 
Ward  and  Dunlop. — Annals  of  Botany,  1887. 
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Myrosins  in  die  Pflanzen.     Journal  fur  Wiss.  Bot.  1893,  XXVI,  p.  55. 
Bulle. — Ann.  de  chim.  et  pharm.,  LXIX,  p.   145. 
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Sur  la  localisation  des  principes  actifs  chez  les  Capparidees.  Comptes 

Rendus,    1893,   587. 
Sur  la  localisation  des  principes  actifs  chez  les  Tropeolees.    Comptes 

Rendus,  1893,  587. 
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CHAPTER  XXII. 

ZYMASE. 

Zymase  or  alcoholic  diastase. — Preparation  of  the  sap  of  yeasts  and  its 
properties. — Determination  of  the  fermenting  power  of  zymase. — 
Chemical  and  physical  conditions  of  the  action  of  zymase. — Experi- 
ment of  Effront  on  intracellular  fermentation. — Industrial  applications 
of  zymase. 

Zymase  or  Alcoholic  Diastase.— The  phenomena  ob- 
served in  alcoholic  fermentation  have  for  a  long  time  occu- 
pied the  scientific  world  and  given  rise  to  numerous  theories 
and  hypotheses. 

In  1858,  Traube  sought  to  explain  the  decomposition  of 
sugar  into  alcohol  and  carbonic  acid  by  the  intervention  of  a 
diastase  secreted  by  the  yeast.  This  opinion  was  accepted 
by  Berthelot  as  well  as  by  some  other  scientists.  None  of 
them,  however,  brought  experimental  proofs  to  show  that 
alcoholic  fermentation  constitutes  a  chemical  reaction  cap- 
able of  being  produced  outside  of  living  cells. 

The  first  attempts  in  this  direction  were  made  in  1871, 
by  Mme.  Manisseim,  who  found  that  the  cells  of  dead  yeast 
can  still  produce,  under  certain  conditions,  a  decomposition 
of  sugar  into  alcohol  and  carbonic  acid. 

The  experiments  of  Mme.  Manassein  were,  however,  far 
from  convincing,  and  did  not  clearly  establish  the  non-inter- 
vention of  cells. 

It  was  Buchner  who,  in  1897,  clearly  showed  the  exist- 
ence, in  the  cells  of  yeast,  of  an  enzyme  causing  alcoholic  fer- 
mentation. By  submitting  yeast  to  a  strong  pressure  he  suc- 
ceeded in  obtaining  a  very  active  liquid  causing  alcoholic  fer- 

276 


ZYMASE.  277 

mentation  in  the  absence  of  any  cells.     He  gave  to  the  en- 
zyme contained  in  this  extract  the  name  of  "  zymase." 

This  discovery  gives  a  definite  explanation  of  alcoholic 
fermentation ;  it  will  certainly  have  a  great  influence  on  the 
study  of  similar  phenomena,  and  will  lead  to  the  discovery  of 
many  other  enzymes.  Once  established  that  alcoholic  fer- 
mentation is  caused  by  a  chemical  substance,  there  is  reason 
to  assume  that  other  similar  phenomena,  such  as  butyric,  vis- 
cous, and  acetic  fermentation,  are  likewise  due  to  diastases 
secreted  by  the  bacteria  producing  these  fermentations.  The 
isolation  of  these  diastases  seems  to  be  only  a  matter  of  time. 

Preparation  and  Properties  of  the  Sap  of  Yeast. — Buch- 
ner  advises  the  following  method  for  the  preparation  of  the 
extract  of  yeast. 

Take  1  kilogram, of  yeast,  to  which  add  1  kilogram  of 
quartz  sand  and  250  grams  of  infusorial  earth.  Crush  the 
mass  to  make  it  plastic  and  pasty.  This  operation  requires 
much  care.  The  crushing  must  be  done  with  a  special 
machine  and  lasts  about  two  hours  per  kilogram  of  yeast. 
The  crushed  mass  is  then  submitted  to  a  pressure  of  500  at- 
mospheres. For  this  purpose  a  hydraulic  press  is  used  and 
the  pressure  should  be  produced  slowly  and  gradually. 

In  this  way  about  320  cubic  centimetres  of  liquid  are  ob- 
tained. The  mass,  from  which  the  sap  has  been  extracted,  is 
ground  with  140  cubic  centimetres  of  water  and  then  pressed 
again  very  slowly  at  500  atmospheres.  Thus  there  is  ob- 
tained, after  2  hours,  180  cubic  centimetres  of  an  extract, 
which  is  added  to  the  liquid  produced  by  the  first  pressure. 
By  this  means,  1  kilogram  of  yeast  furnishes  500  cubic  cen- 
timetres of  extract.  The  liquid  is  stirred  with  4  grams  of 
infusorial  earth,  filtered  through  paper,  and  poured  into  a 
cooled  receptacle. 

The  extract  obtained  by  Buchner's  process  is  clear,  light 
yellow,  and  has  a  characteristic  odor.  According  to  the 
origin  of  the  yeast  which  has  been  used  in  its  preparation,  the 
liquid  contains  from  7  to  10  per  cent  of  dry  substance. 


278  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

The  analysis  of  the  liquid  shows  the  following  figures : 

Dry  substance .  .  .  .      6.7 

Ash 1. 15 

Albuminoid  substances 3.7 

In  this  analysis  the  albuminoid  substances  are  calculated 
according  to  the  richness  of  the  liquid  in  nitrogen. 

The  extract  of  yeast  is  saturated  with  carbonic  acid  and 
when  it  is  brought  to  the  boiling-point,  one  observes  an  abun- 
dant liberation  of  this  gas  and  a  strong  coagulation  which 
gives  the  liquid  a  semi-solid  aspect. 

This  liquid  acts  differently  towards  different  sugars ;  lac- 
tose and  mannite  remain  intact  in  presence  of  the  extract  as 
in  presence  of  yeast-cells ;  saccharose,  dextrose,  levulose, 
and  maltose,  mixed  with  an  equal  amount  of  the  yeast  ex- 
tract, at  the  end  of  a  quarter  of  an  hour  disengage  carbonic 
acid,  an  action  which  sometimes  lasts  several  days. 

The  fermenting  power  of  the  liquid  persists  after  it  has 
been  through  a  Berkefeld  filter ;  the  activity  of  the  liquid  is 
not  destroyed  by  passage  through  the  Chamberland  filter, 
but  the  fermenting  power  weakens,  however,  to  a  greater 
degree  than  in  passing  through  the  Berkefeld  filter.  The 
fermentation  is  retarded  by  these  operations  :  the  extract  fil- 
tered in  a  Berkefeld  filter  produced  fermentation  only  at  the 
end  of  a  day. 

The  active  substance  contained  in  the  extract  is  capable 
of  diffusing  through  dialyser  paper ;  in  fact,  when  a  dialyser 
containing  a  certain  quantity  of  yeast  extract  is  placed  in  a 
37  per  cent  solution  of  saccharose,  numerous  bubbles  of  car- 
bonic acd  are  seen  to  appear  at  the  surface  of  the  sugar  solu- 
tion. 

Yeast  extract  can  be  dried  at  30°-35°  without  losing  its 
activity.  By  drying  in  vacuo,  a  hard  product  is  obtained  pre- 
senting the  appearance  of  white  of  egg.  A  filtered  solu- 
tion of  this  product  possesses  the  same  properties  as  yeast 
extract;  in  a  dry  state  it  keeps  several  months. 


ZYMASE.  279-. 

For  the  preparation  of  concentrated  extract  of  yeast,  one 
proceeds  in  the  following  manner :  500  cubic  centimetres  of 
sap  are  evaporated  in  vacuo  at  200  or  25 °  to  a  syrupy  con- 
sistency. The  evaporation  must  be  done  very  rapidly  and 
lasts  about  half  an  hour.  The  syrup  obtained  is  then  spread 
in  thin  layers  on  glass  plates  and  replaced  in  vacuo,  or  else 
left  in  the  air  at  a  temperature  of  30°  or  35 °  so  that  it  can 
evaporate.  After  24  hours  the  dried  substance  is  scraped 
from  the  glass,  reduced  to  powder  and  completely  dried  over 
sulphuric  acid.  Five  hundred  grams  of  yeast  extract  furnish 
70  grams  of  a  very  soluble  powder  which  shows  great  ac- 
tivity. 

It  is  to  be  observed  that  the  concentrated  extract  of  the 
juice  keeps  much  better  than  the  diluted  extract.  The  solu- 
tion of  diluted  extract  is  rapidly  destroyed  in  the  presence  of 
oxygen,  while  this  same  solution,  brought  to  a  syrupy  con- 
sistency, keeps  for  a  very  long  time,  even  at  a  temperature  of 
300  and  in  the  air. 

Buchner  succeeded  in  separating  the  diastase  from  the 
yeast  sap  by  adding  to  the  latter  12  times  its  volume  of  abso- 
lute alcohol. 

The  precipitate  thus  obtained,  and  dried,  is  a  white  powder 
having  the  same  properties  as  the  extract  but  possessing  a 
very  slight  fermenting  power. 

The  zymase  enclosed  in  the  cells  resists  quite  high  tem- 
peratures. A  yeast  dried  in  the  air  at  a  temperature  of  370 
and  then  heated  to  ioo°  for  6  hours  is  still  capable  of  pro- 
ducing alcoholic  fermentation  in  a  solution  of  saccharose. 
The  cells  of  yeast  which  could  not  resist  this  temperature  are 
killed  and  no  longer  reproduce. 

If,  instead  of  bringing  it  to  ioo°,  the  yeast  is  brought  to  a 
temperature  of  I40°-I45°,  the  cells  lose  all  fermenting 
power.  Zymase  is  then  more  resistant  to  the  action  of  heat 
than  the  cells  which  secrete  it. 

Determination  of  the  Fermenting  Power  of  Zymase. 
— The  fermenting  power  cf  yeast  is  measured  by  a  method 


280  THE  ENZYMES  AND    THEIR   APPLICATIONS. 

recommended  by  Meissel  for  the  determination  of  the  alcohol 
in  fermented  solutions.  This  method  is  based  on  the  meas- 
urement of  the  carbonic  acid  formed  during  the  fermentation. 

Forty  cubic  centimetres  of  extract  are  introduced  into  a 
flask  of  1 20  cubic  centimetres  capacity,  and  a  quantity  of 
powdered  saccharose  sufficient  to  obtain  a  12  to  15  per  cent 
sugar  solution  is  added.  The  flask  is  left  alone  for  a  few 
minutes,  after  which  it  is  shaken  and  closed  with  a  rubber 
stopper  through  which  two  tubes  are  passed.  One  of  them 
is  furnished  with  a  tap  on  the  outside,  and  descends  to  the 
surface  of  the  liquid.  The  other  tube  is  open  and  communi- 
cates with  a  washing-bottle  containing  2  cubic  centimetres  of 
sulphuric  acid;  the  open  end  is  provided  with  a  rubber 
Bunsen  valve. 

At  the  end  of  the  experiment  the  tap  is  opened,  air  is  al- 
lowed to  pass  into  the  apparatus  so  as  to  drive  out  the 
carbonic  acid  and  the  apparatus  is  placed  on  the  balance. 
The  difference  in  weight  shows  the  quantity  of  carbonic  acid 
disengaged. 

Method  of  Decomposition  of  Sugar  by  Zymase. — A  solu- 
tion of  cane-sugar  with  arseniate  of  potassium  added  and  fer- 
mented with  450  cubic  centimetres  of  yeast  extract  at  120  for 
40  hours  furnished  6.67  gr.  of  carbonic  acid  and  J.J2  gr.  of 
alcohol,  the  alcohol  which  was  in  the  extract  at  the  beginning 
being  deducted. 

In  fermentation  produced  by  yeast-cells  there  is  theoret- 
ically obtained  48.89  parts  of  carbonic  acid  and  51.1 1  parts  of 
alcohol.  By  comparing  these  figures  with  the  preceding, 
Buchner  found  that  the  relation  between  the  quantities  of 
carbonic  acid  and  alcohol  is  practically  the  same  in  the  two 
cases,  and  that  the  decomposition  of  sugar  by  yeast  extract 
is  accomplished  in  the  same  way  as  by  the  cells. 

It  appears  from  these  data  that  the  fermenting  power  of 
yeast  extract  can  be  measured  by  the  carbonic  acid  liberated 
during  its  action.  These  experiments  of  Buchner  show  us 
only  the  general  lines  of  the  course  of  decomposition  of  sugar 


ZYMASE. 


by  zymase.  The  method  he  adopted  for  the  determination 
of  alcohol  and  carbonic  acid  is  far  from  being  exact  and  from 
giving  information  on  the  degree  of  purity  of  the  fermenta- 
tion obtained  with  zymase.  Pasteur  demonstrated  that  the 
entire  amount  of  sugar  which  disappears  during  fermentation 
is  not  transformed  quite  in  accordance  with  the  following 
equation : 

C6H12Oe  =  2C02  +  2C2H5OH. 

There  is  always  a  little  sugar  which  is  broken  up  in  a  dif- 
ferent way,  and  furnishes  glycerin  and  succinic  acid.      It  is 
very  probable  that  zymase  acts  quite  differently  from  beer- 
yeasts,  and  that  the  fermentation  brought  about  by  its  use 
may  give  much  purer  products  than  those  obtained  by  yeasts. 
Still,  the  zymase  isolated  from  the  cells  is  relatively  weak 
one  hundred  cubic  centimetres  of  this  extract,  representing 
200  grams  of  yeast,  produce,  by  acting  on  a  sugar  solution 
less  carbonic  acid  than  a  gram  of  yeast.      Probably  there 
exists   in   the  yeast-cells   only  a   small   quantity   of  zymase 
which,  further,  undergoes  a  change  during  extraction. 

Influence  of  Physical  and  Chemical  Conditions.— Zymase 
is  very  sensitive  to  the  action  of  the  temperature  A  27 
per  cent  solution  of  sugar,  with  yeast  sap  added,  produces 
very  different  quantities  of  carbonic  acid  according  to  the 
temperature  of  fermentation. 

Temperature.  Carbonic  acid  (in  grams)  formed  after 

6hours-      21  hours.     24~hours.     40  hours.' 

14 °-43         1. 11         1. 14         1.27 

22° °76         I.OI  1.02         1.00 

QAt  the  beginning  of  the  experiment  the  temperature  of 
22  is  very  favorable  to  the  action  of  the  enzyme;  after  6 
hours  of  fermentation  there  is  found,  at  this  temperature  a 
liberation  of  0.76  grams  of  carbonic  acid,  while  at  120  to  i> 
there  is  formed  only  0.43  gr.  of  this  gas.  But  if  the  opera- 
tion   is    prolonged    at    a    temperature    of   22°,    the    action 


282  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

slackens;  this  slackening  is  evidently  because  of  a  partial 
destruction  of  the  diastase. 

The  course  of  fermentation  is  also  influenced  by  the  con- 
centration of  the  sugar  solutions. 

CO»  (in  grams)  after 
Saccharose, , 

per  cent.                        i6  hours>  2^  hours.  40  hours. 

l6     1-33  I.46  I.48 

27     O.7O  O.80  O.82 

37     O.60  O.72  O.74 

In  liquids  containing  16  per  cent  of  sugar  the  fermenta- 
tion is  more  active  than  in  those  which  contain  27  per  cent, 
but  the  enzyme  still  acts  on  solutions  containing  37  per  cent 
of  sugar.  In  the  solutions  containing  from  40  to  50  per  cent 
of  sugar  the  fermentation  is  almost  completely  stopped,  but 
the  enzyme  is  not  at  all  changed,  for,  by  diluting  the  solution, 
fermentation  can  be  brought  about  anew. 

The  diastatic  activity  of  zymase  diminishes  in  proportion 
as  its  action  is  prolonged ;  if  we  compare  the  quantity  of  car- 
bonic acid  formed  by  the  action  of  the  enzymes  during  the 
first  16  hours  with  the  quantity  of  gas  freed  during  the  fol- 
lowing 16  hours,  we  find  a  rapid  decrease  of  the  fermenting 
power.  By  calculating  this  power  per  100  cubic  centimetres 
of  extract  per  hour,  Buchner  found  that  zymase  furnishes,  on 
the  average,  the  following  quantities  of  carbonic  acid: 

From  1  to  16  h.  16  to  24  h.  24  to  40  h.  40  to  60  h. 

Average  of  3  experiments.  . .     0.17  gr.  0.060  gr.  0.020  gr.        0.002  gr. 

"         "2  "  ■-.     0.11    "  0.010    "  0.002    " 

"  "2  "  ...     0.08    "  0.016    "  0.004    " 

Yeast  extract,  like  all  diastatic  solutions,  produces  in 
"hydrogen  peroxide  a  liberation  of  oxygen. 

When  hydrocyanic  acid  has  been  added  to  the  extract  it 
loses  this  characteristic  property.  But  if  the  extract  with  hy- 
drocyanic acid  added  is  then  submitted  to  prolonged  action 
of  the  air,  the  reaction  with  hydrogen  peroxide  reappears. 


ZYMASE.  283 

It  is,  therefore,  probable  that  hydrocyanic  acid  combines 
with  the  diastases,  forming  a  combination  which  impedes 
their  activity,  and  that  this  combination  is  destroyed  by  con- 
tact with  the  oxygen  which  regenerates  the  enzyme. 

An  experiment  of  Buchner  relative  to  the  action  of  hy- 
drocyanic acid  and  air  on  zymase  may  be  given  : 

Four  cubic  centimetres  of  extract  were  mixed  with  6 
cubic  centimetres  of  2  per  cent  hydrocyanic  acid;  to  one  half 
(A)  of  the  mixture  were  added  3  grams  of  cane-sugar;  the 
other  half  (B)  was  submitted  to  the  action  of  the  air  for  1 
hour,  then  3  grams  of  saccharose  were  added.  The  liquids 
were  placed  in  U  tubes  closed  at  one  end.  In  experiment  A 
not  a  trace  of  carbonic  acid  was  produced,  while  in  experi- 
ment B  the  closed  part  of  the  tube  was  filled  with  this  gas  at 
the  end  of  20  hours,  the  liberation  having  begun  at  the  end 
of  5  hours. 

Zymase  is  influenced  by  the  chemical  conditions  of  the 
medium. 

Neutral  salts,  like  ammonium  sulphate,  calcium  chloride, 
etc.,  have  a  retarding  influence  on  fermentation. 

A  direct  relation  is  also  found  to  exist  between  the  ac- 
tivity of  the  yeast  extract  and  the  presence  of  coagulable 
albumen  in  the  extract. 

Yeast  extract,  kept  for  some  time  at  a  temperature  of  350 
to  400,  becomes  turbid,  contains  flocculent  masses,  and  loses 
its  activity.  On  the  other  hand,  it  has  been  observed  that 
when  an  extract  becomes  inactive  for  any  reason,  it  scarcely 
coagulates  at  all  at  a  temperature  of  400  to  500.  This  rela- 
tion between  the  presence  of  coagulable  materials  and  the 
diastatic  activity  is  explained  by  Buchner  in  the  following 
way.  According  to  him,  zymase  is  an  albuminoid  substance 
which  is  coagulated  by  heat,  but  this  coagulation  does  not 
occur  when  the  diastatic  substance  is  transformed. 

Buchner  also  interprets  the  great  instability  of  zymase  by 
the  presence  of  a  peptonizing  diastase  in  the  yeast  extract; 
this  enzyme  would  act  on  zymase  rendering  it  inactive.     It 


284  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

is  the  simultaneous  presence  of  these  two  ferments,  the  one 
peptonizing  and  the  other  producing  the  decomposition  of 
sugar,  which  explains  the  activity  of  yeast  extract  at  rela- 
tively low  temperatures. 

At  220  peptase  acts  with  more  energy  than  zymase,  while 
at  a  low  temperature  the  peptonizing  action  is  not  complete 
and  zymase  can  produce  greater  quantities  of  alcohol. 

The  favorable  influence  of  sugars  on  the  preservation  of 
zymase  also  speaks  in  favor  of  the  hypothesis  of  the  digestion 
of  zymase  by  the  peptonizing  enzyme. 

It  is  known  that  in  concentrated  solutions  of  saccharose 
the  digestion  of  fibrin  by  pepsin  is  retarded.  Now,  if  one 
volume  of  yeast  extract  is  mixed  with  one  volume  of  a  75  per 
cent  solution  of  saccharose,  a  solution  is  obtained  which 
keeps  for  a  week  at  the  ordinary  temperature  and  for  15  days 
in  a  refrigerator.  Sugar,  therefore,  has  a  very  favorable  ac- 
tion on  the  preservation  of  zymase. 

The  activity  of  yeast  extract  varies  noticeably  according 
to  the  variety  of  yeast  used  in  its  preparation.  Generally 
bottom  yeasts  give  a  very  active  extract,  while  bakery  yeasts 
contain  hardly  any  zymase.  A  great  difference  is  also  ob- 
served between  extracts  of  fresh  yeasts  and  those  of  yeasts 
which  have  remained  in  the  air  for  some  time.  The  latter 
give  less  active  extracts.  However,  not  all  brewery  yeasts 
give  an  extract  of  the  same  activity.  The  origin  of  the  yeast 
plays  an  important  part  here.  The  differences  observable  ac- 
cording to  the  origin  and  age  of  the  yeast  bring  up  an  argu- 
ment for  the  hypothesis  explaining  the  alteration  of  zymase 
by  the  action  of  another  enzyme. 

Buchner  divided  a  certain  quantity  of  yeast  into  two  parts 
A  and  B.  The  extract  was  pressed  out  from  A  immediately, 
while  the  portion  B  was  left  to  itself  for  3  days  at  7°-8°  be- 
fore the  sap  was  expressed.  The  extract  of  A  possessed  a 
strong  activity,  while  the  liquid  coming  from  B  gave  only  a 
small  quantity  of  carbonic  acid.  Buchner  attributed  the 
diminution  of  activity  in  the  second  case  to  a  peptonization. 


ZYMASE.  285 

produced  during  the  3  days'  standing  at  7°-8°.  The  action 
of  pepsin  would  also,  according  to  him,  be  the  cause  of  altera- 
tion of  the  diastase  contained  in  compressed  yeast. 

This  opinion  is  also  reinforced  by  an  experiment  of  Hohn, 
who  quite  recently  demonstrated  the  presence  of  the  pro- 
teolytic enzyme  in  the  sap  of  yeasts. 

Buchner  has,  however,  shown  directly  the  peptonization 
of  zymase  by  the  enzyme  by  the  following  experiment: 

He  placed  on  ice  3  tubes  containing  3  cubic  centimetres 
of  yeast  extract.  To  two  of  these  tubes  were  added  0.1  gr. 
of  trypsin,  the  third  serving  as  the  control.  After  12  hours 
each  tube  received  2  grams  of  pulverized  saccharose.  The 
experiments  made  with  trypsin  remained  absolutely  inactive 
after  the  addition  of  cane-sugar;  on  the  other  hand,  in  the 
control  tube  a  very  active  fermentation  was  produced. 

The  discovery  of  Buchner  has  had  numerous  opponents, 
who  have  tried  to  show  that  yeast  extract  always  contains 
either  cells  or  ferments  and  who  have  attributed  the  decom- 
position of  sugar  to  the  intervention  of  living  cells. 

Buchner  successfully  refuted  all  these  objections  by 
means  of  conclusive  experiments. 

The  existence  of  zymase  is  demonstrated  by  the  following 
facts : 

1st.  An  alcoholic  fermentation  can  be  obtained  with  the 
solid  substance  obtained  by  the  precipitation  of  the  sap  by 
alcohol ; 

2nd.  With  yeast  extract  an  almost  instantaneous  fermen- 
tation may  be  obtained,  whose  intensity  diminishes  with  time. 
If  it  is  a  question  of  living  cells  a  contrary  phenomenon  is 
observed :  the  fermentation  increases  in  intensity  as  the  cells 
develop ; 

3rd.  Yeast  extract  produces  a  fermentation  in  the  pres- 
ence of  amounts  of  antiseptics  which  check  the  activity  of 
living  cells ; 

4th.  By  passage  through  a  porcelain  filter,  active  liquids 


2  86  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

are  obtained  without  it  being  possible  to  discern  the  pres- 
ence of  organisms. 

To  sum  up,  alcoholic  fermentation  is  produced  by  chem- 
ical agents,  without,  and  in  the  absence  of,  living  cells. 

It  is  true  that  the  material  which  produces  this  trans- 
formation is  elaborated  by  the  vital  activity,  and  that  its 
formation  is  intimately  related  to  the  growth  and  multiplica- 
tion of  the  cells.  The  fermenting  power  of  the  cells  is,  there- 
fore, reduced  to  their  ability  to  produce  zymase. 

Intracellular  Fermentation.  — Zymase  should  be  found 
in  many  other  living  cells.  The  fermenting  power  which  can 
develop  in  certain  fungi  should,  it  seems  to  us,  be  attributed 
to  a  secretion  of  zymase  which  occurs  under  special  con- 
ditions. 

There  is  also  reason  to  believe  that,  in  the  phenomena  of 
intracellular  fermentation,  it  is  zymase  again  which  plays  an 
•active  part.  Pasteur  found  that  fruits  plunged  in  carbon- 
dioxide  gas  enter  into  fermentation  and  transform  sugar  into 
alcohol  and  carbonic  anhydride. 

Muntz,  by  replacing  air  by  nitrogen,  found  the  same 
phenomenon  for  leafy  plants.  Under  these  conditions  al- 
cohol is  formed  in  the  leaves  of  the  plant.  This  phenomenon 
is  explained  by  vital  activity  and  it  is  assumed  that  the 
changes  in  the  work  are  due  to  changes  in  the  conditions  of 
nutrition. 

It  is  easier,  we  believe,  to  suppose  that  the  absence  of 
oxygen  is,  under  these  conditions,  favorable  to  the  secretion 
of  zymase.  The  fermentation  observed  in  this  case  is  similar 
to  that  produced  by  the  action  of  alcoholic  yeast;  in  both 
cases  the  zymase  is  the  cause. 

The  action  of  zymase  in  fruits  protected  from  the  air 
has  furnished  us  with  the  subject  of  interesting  researches 
which  we  are  at  present  pursuing,  and  which  are  still  far 
from  being  completed;  but  we  can  even  now  give  some 
information  which  will  find  its  complete  development  in  a 
later  work. 


ZYMASE.  287 

The  numerous  experiments  we  have  made  have  confirmed, 
in  our  opinion,  the  presence  of  zymase  in  fruits,  especially 
cherries  and  plums,  in  peas,  and  in  barley. 

The  first  experiments  were  made  with  cherries  in  the  fol- 
lowing way : 

The  fresh  fruit  was  washed  in  a  dilute  solution  of  formic 
aldehyde  to  destroy  the  micro-organisms,  then  carefully 
wiped  and  submerged  in  flasks  containing  olive  oil.  At  the 
end  of  3  days  the  cherries  were  covered  with  little  bubbles  of 
gas  and  there  was  then  found,  above  the  oil  which  covered 
the  fruits,  a  liberation  of  carbonic  acid  which  increased  after 
the  5th  day. 

Fermentation  continued  very  slowly  for  20  days  at  a  tem- 
perature of  io°.  After  this  time  the  oil  was  poured  off,  the 
cherries,  and  also  the  stones,  were  crushed  in  a  mortar  and 
the  juice  pressed  out  by  squeezing  the  mass  in  a  cloth. 

The  residue  was  removed  from  the  cloth,  treated  cold 
with  ether  to  free  it  from  the  oil,  then  dried  in  vacuo,  and  re- 
duced to  a  fine  powder,  which  was  soaked  in  2  volumes  of 
water  with  a  small  amount  of  ether  added.  It  was  left  in  a 
corked  bottle  at  50  for  12  hours,  after  which  the  mass  was 
submitted  to  strong  pressure.  Thus  a  liquid  was  obtained 
which,  filtered  through  filter-paper,  wras  a  viscous,  trans- 
parent solution,  of  slightly  acid  reaction,  and  giving  the  re- 
actions of  guaiacum  and  hydrogen  peroxide. 

The  presence  of  zymase  in  such  a  liquid  may  be  found  by 
the  following  experiments : 

To  50  cubic  centimetres  of  the  liquid  add  7  grams  of  pow- 
dered cane-sugar  and  leave  for  6  hours  at  220  in  a  small  flask 
furnished  with  a  delivery-tube.  After  2  hours,  the  formation 
of  carbonic  acid  is  observed,  and  after  6  hours  a  diminution 
in  weight  of  3  decigrams. 

A  parallel  experiment  is  made  with  the  same  liquid  pre- 
viously kept  at  400  for  an  hour  in  a  closed  flask,  then  cooled 
to  22°  and  left  at  this  temperature  for  5  hours,  leaving  the 
delivery-tube  open.    In  this  second  experiment  neither  liber- 


288  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

ation  of  gas  nor  diminution  of  weight  is  found.     The  alco- 
holic diastase  has  therefore  been  destroyed  by  heat. 

.  The  analysis  of  the  unfermented  sugar  solution  shows 
that  a  change  has  occured  in  its  chemical  composition.  We 
have,  for  example,  found  in  one  of  our  experiments  that 
3.4  grams  of  saccharose  had  been  transformed  into  invert- 
sugar. 

This  transformation  cannot  be  attributed  to  the  acidity 
of  the  medium,  for  by  heating  the  active  liquid  in  a  closed 
vessel  for  10  minutes  at  8o°  before  the  addition  of  sugar,  and 
by  then  keeping  the  liquid  with  sugar  added  for  an  hour 
at  400,  then  for  5  hours  at  220;  we  obtained  only  0.15  gr.  of 
invert-sugar  instead  of  3.4  gr.  The  active  liquid  evidently 
contains  zymase  and  sucrase,  and  while  the  zymase  is  de- 
stroyed by  the  heat,  the  sucrase  is  not  changed. 

The  existence  of  zymase  in  the  juice  of  cherries  has  been 
confirmed  by  other  experiments  in  which  it  has  been  pos- 
sible to  measure  the  alcohol  produced.  The  following 
method  of  procedure  was  employed: 

To  200  cubic  centimetres  of  active  juice  were  added  sugar 
and  2  grams  of  chloroform.  In  a  parallel  experiment,  a  little 
chloroform  and  2  grams  of  yeast  were  added  to  a  15  per  cent 
solution  of  sugar.  The  two  liquids,  left  5  days  at  io°,  gave 
.different  results. 

In  the  solution  containing  yeasts  fermentation  did  not 
take  place,  while  in  the  juice  of  the  cherries  0.8  gr.  of  alcohol 
was  found. 

The  non-existence  of  yeasts  in  the  fermented  solution 
was  confirmed  by  microscopic  analysis  as  well  as  by  cultiva- 
tion on  plates. 

Experiments  made  with  a  view  to  precipitating  the  dia- 
stase by  alcohol  have  not  furnished  satisfactory  results.  The 
active  liquid  loses  its  properties  in  passing  through  porce- 
lain bougies,  and  the  zymase  which  we  obtained  differs  in 
this  respect  from  the  enzyme  isolated  by  Buchner. 

In  the  course  of  our  experiments  we  found,  furthermore, 


ZYMASE.  289 

that  fresh  peas,  as  well  as  barley,  furnish  quite  considerable 
quantities  of  alcohol  by  intracellular  fermentation.  Sugar- 
peas,  left  in  oil,  gave  on  analysis  2  per  cent  of  alcohol.  Bar- 
ley, previously  soaked,  dried,  and  put  in  oil,  gave  1.6  per  cent 
of  alcohol. 

By  treating  these  seeds  by  processes  similar  to  those 
used  for  cherries,  we  were  able  to  discover  the  presence  of 
zymase. 

Industrial  Applications  of  Zymase. — Zymase,  while 
very  interesting  from  a  theoretical  point  of  view,  will  per- 
haps also  have  in  the  future  numerous  industrial  applica- 
tions. 

The  fermentation  produced  by  the  enzyme,  without 
direct  intervention  of  yeasts,  presents  theoretically  a  great 
advantage.  In  this  way  much  more  rapid  fermentations 
can  be  obtained,  and  purer  and  better  products. 

At  the  present  time  distillers  and  brewers  cultivate  their 
own  yeasts  and  seek  to  adapt  them  to  their  kind  of  work. 
However,  even  starting  with  pure  yeasts,  good  results  are 
not  always  obtained;  often  infection  takes  place  and  con- 
sequently a  degeneration  of  the  yeast. 

It  is  to  be  hoped  that  in  the  fuUire  the  cultivation  of 
yeasts  and  the  subsequent  preparation  of  zymase  will  be 
done  in  special  manufactories,  where  the  brewers  and  dis- 
tillers will  produce  preparations  of  great  activity  and  pro- 
ducing an  immediate  action. 

It  is  true  that  in  the  brewery  yeast  plays  also  an  im- 
portant part  from  the  point  of  view  of  the  elimination  of 
nitrogenous  materials,  a  work  which  zymase  alone  could  not 
do.  It  would  be  necessary,  therefore,  in  working  with  en- 
zymes alone,  to  change  completely  the  technique  and  invent 
new  processes. 

The  discovery  of  zymase  is  too  recent  to  have  caused 
great  industrial  changes  immediately;  however,  the  first  at- 
tempts at  practical  application  have  already  been  made  by 
Buchner,  who  has  invented  a  new  process  having  for  its  aim 


290  THE  ENZYMES  AND    THEIR   APPLICATIONS. 

the  preparation  of  a  durable  yeast  intended  to  replace 
pressed  yeast  in  bread-making. 

This  process  consists  in  first  drying  the  yeast  at  low  tem- 
perature, then  heating  to  500,  then  to  ioo°,  and  then,  when 
the  yeast  is  completely  dried,  grinding  it  to  powder.  It 
is  then  ready  for  the  market. 

This  process  has  many  advantages.  The  yeast  pre- 
pared in  this  way  keeps  much  better  than  compressed  yeast, 
dead  cells  being  less  subject  to  change  than  living  cells. 
Moreover,  the  greater  drying  of  this  yeast  recommends  it 
from  a  hygienic  point  of  view,  for  the  micro-organisms  which 
yeast  breads  always  contain,  are  here  destroyed  and  can  no 
longer  affect  the  dough,  even  when  it  is  insufficiently  steri- 
lized. 

Buchner's  yeast  is  called  enduring  yeast  (Dauerhefe) ; 
it  is  used  in  the  bakery  in  the  same  way  as  the  ordinary 
compressed  yeasts.  According  to  the  author  of  the  patent, 
experiments  have  proved  that  the  addition  of  5  to  10  per 
cent  of  enduring  yeast  are  sufficient  to  produce  a  satisfactory 
dough. 

BIBLIOGRAPHY. 

Berthelot. — Chimie  organique  fondee  par  la  synthese. 

E.  Buchner. — Alkoholische  Gahrung  ohne  Hefezellen.  Berichte  der 
deutsch.  chem.  Gesellschaft,  XXX,   1897,  p.   117. 

Ueber  zellenfreie  Gahrung.  Berichte  der  deutsch.  chem.  Gesell- 
schaft, XXX,  1 1 10. 

E.  Buchner. — Procede  pour  la  fabrication  des  levures  d'attente.  Brevet, 
Allemagne,  1897,  2668,  n°  97240. 

Bechamp. — Sur  la  presence  de  l'alcool  dans  les  tissus  animaux  pendant 
la  vie  et  apres  la  mort,  dans  les  cas  de  putrefaction,  aux  points  de  vue 
physiologique  et  toxicologique.     Comptes  Rendus,  1879,  p.  573. 

Hahn. — Berichte  der  deutsch.  chem.  Gesellschaft,  1898. 

Geret  and  Hahn. — Berichte  der  deutsch.  chem.  Gesellschaft,  1898. 

Pasteur. — Etude  sur  la  biere.     Comptes  Rendus,  1876,  LXXVII,  p.  1140. 

Etude  sur  la  biere,  1876.     Paris,  Gauthier-Villars. 

Memoires  sur  la  fermentation  alcoolique.     Comptes  Rendus,  1859,  P- 

1149,  XIV,  III.     Bull,  de  la  Soc.  de  chim.     Paris,  1861. 

Sur  la  production  de  l'alcool  par  les  fruits.     Comptes  Rendus,  1872,. 

p.  1054,  LXXXV. 


ZYMASE.  291 

Pasteur. — Sur  la  theorie  de  la  fermentation.     Comptes  Rendus,  LXXV. 
Liebig. — Sur  les  phenomenes  de  fermentation  et  de  putrefaction.     Ann. 

de  chim.  et  de  phys.,  1889,  t.  LXXI,  p.  147. 
A.    Munz. — De    la    matiere    sucree    contenue    dans    les    champignons. 

Comptes  Rendus,  1874,  t.  LXXIV. 
Recherches   sur  la  fermentation   alcoolique   intracellulaire    dans   les 

vegetaux.     Comptes  Rendus,  1878,  t.  LXXXVI,  p.  49. 
Recherches   sur  la  fermentation  intracellulaire  des  vegetaux.     Ann. 

de  chim.  et  de  phys.,  1878,  5e  serie,  t.  XIII,  p.  543. 

Alcools  du  sol.     Comptes  Rendus,  XCII,  p.  499. 

Neumeister. — Ber.  der  deutschen  chemischen  Gesellschaft,  XXX,  p.  2963. 
Stavenhagen. — Ber.  der  deutschen  chemisch.  Gesellschaft,  XXX,  p.  2422. 
Marie  Manassein. — Ber.  der  deutschen  chem.  Gesellschaft,  XXX,  p.  3061. 
E.  Buchner  and  Rapp. — Alkoholische  Gahrung  ohne  Hefezellen.  Berichte 

der  deutsch.  chem.  Gesellschaft,  1897,  3,  2670;  1898,  1,  209;  1898,  i, 

1084;  1898,  1,  109. 


CHAPTER  XXIII. 

OXIDASES. 

Presence  of  oxidases  in  vegetable  and  animal  cells. — General  properties. — 
Laccase. — Tyrosinase. — Influence  of  the  medium. — Action  of  oxidases 
on  phenols  insoluble  in  water. — The  "breaking"  of  wines:  cenoxi- 
dase. — Oxidin. — Olease. 

Soluble  ferments  have  for  a  long  time  been  considered 
as  substances  acting-  only  as  hydrolyzing  agents,  that  is 
causing  the  fixation  of  one  or  more  molecules  of  water  at  the 
same  time  with  a  molecular  decomposition.  Oxidation  and 
dehydration,  molecular  change  without  fixation  of  water,  all 
these  chemical  phenomena  were  attributed  to  the  direct  ac- 
tion of  vital  energy  without  any  diastatic  intervention. 

This  entirely  erroneous  theory  has  lately  been  success- 
fully refuted  by  a  series  of  discoveries  accomplished  in  the 
domain  of  biological  chemistry  by  Bertrand,  Bourquelot, 
Hikorokuro  Yoshida,  Cazeneuve,  Martinand,  etc.,  whose 
works  we  shall  have  occasion  to  examine. 

The  studies  of  these  scientists  have  demonstrated  the 
existence  of  a  series  of  substances  having  characteristics  of 
true  oxidizing  agents,  and  causing  oxygen  to  unite  with 
certain  bodies.  These  substances,  secreted  by  living  cells, 
have  received  the  name  of  oxidases.  These  enzymes  facili- 
tate the  oxidation  of  certain  substances,  either  by  dehydrat- 
ing them  or  by  enlarging  their  molecule  by  the  addition  of 
oxygen. 

Certain  vegetable  juices,  such  as  wine,  the  latex  of  the 
lac-tree,  the  juice  of  pears,  plums,  and  other  fruits,  as  well  as 

292 


OXIDASES.  293 

oertain  fungi,  change  when  exposed  for  some  time  to  the  air. 
This  phenomenon,  which  is  generally  shown  by  a  change 
of  color  or,  in  case  of  solid  bodies,  by  an  increase  in  tem- 
perature, does  not  occur  in  vacuo.  It  therefore  possesses 
all  the  characteristics  of  oxidation,  because  the  intervention 
of  air,  and  consequently  of  oxygen,  is  indispensable  to  its 
production. 

The  direct  cause  of  this  oxidation  remained  for  a  long 
time  unknown.  It  was  discovered  in  1883  by  a  Japanese 
chemist,  Hikorokuro  Yoshida,  who,  by  making  experiments 
on  the  oxidation  of  the  latex  of  the  lac-tree,  discovered  in 
this  phenomenon  the  intervention  of  a  diastase. 

This  discovery  gave  a  new  impetus  to  studies  upon  en- 
zymes ;  the  question  was  taken  up  in  different  laboratories, 
and  discoveries  bearing  on  the  most  varied  subjects  were 
soon  numerous  and  conclusive.  There  were  successively 
studied  vegetable  tissues,  muscles,  organic  secretions,  and 
each  investigation  carried  on  in  this  field  brought  new  proofs 
of  the  existence  of  oxidases. 

The  oxidation  of  the  latex  of  the  lac-tree  and  its  transfor- 
mation into  a  black  varnish  was  clearly  recognized  as  a 
phenomenon  of  the  diastatic  order.  A  transformation  pre- 
senting some  similar  characteristics  and  occurring  in  the 
juices  of  numerous  vegetables,  such  as  mushrooms,  pota- 
toes, beets,  and  the  rhizomes  of  Canna.indica,  was  attributed 
by  Bourquelot,  Lindet,  Bertrand,  etc.,  to  another  oxidizing 
enzyme. 

The  decoloration  of  wine  and  the  deposition  of  the  color- 
ing matter  were  recognized  as  being  phenomena  of  the  same 
order,  and  were  attributed  to  the  action  of  a  diastase  which 
certain  authors  regard  as  pre-existent  in  the  must,  while 
others  consider  it  as  the  product  of  a  mould :  Botrytis  cinerea. 

In  the  animal  kingdom  the  experimenters  had  occasion 
to  make  quite  as  numerous  and  quite  as  interesting  dis- 
coveries. 

An  oxidizing  ferment  was  found  in  the  saliva  as  well 


294  THE  ENZYMES  AND    THEIR   APPLICATIONS. 

as  in  other  secretions — nasal  mucus,  tears,  semen — while  the 
urine,  bile,  and  intestinal  secretions  were  found  to  be  free 
from  any  ferment  of  the  kind. 

Jacquet,  in  1882,  made  experiments  on  the  oxidation  of 
benzyl  alcohol  and  salicylic  aldehyde  with  pieces  of  lungs, 
loins,  and  muscles  of  the  horse,  previously  treated  with  car- 
bolised  water,  then  frozen  and  reduced  to  pulp.  These  frag- 
ments of  organs  caused  an  oxidation  which  was  no  longer 
produced  when  they  had  been  cooked  in  boiling  water. 

Even  at  that  time  Jacquet  realized  that  the  oxidation  did 
not  come  solely  from  the  cells,  because  the  aqueous  extract 
of  these  tissues,  as  well  as  the  cells  themselves,  produced  a 
fixation  of  oxygen  on  benzyl  alcohol  and  salicylic  aldehyde. 

Aleloos  and  Brauwer  confirmed  these  results  by  collect- 
ing a  substance,  extracted  from  a  horse's  liver,  which,  pre- 
cipitated from  its  aqueous  solution  by  alcohol,  oxidized 
formic  aldehyde  and  transformed  it  into  acid  with  liberation 
of  carbon  dioxide.  This  substance  lost,  moreover,  all 
oxidizing  action  after  having  been  heated  to  ioo°. 

Spitzer  and  Rhomann  found  this  substance  in  the  blood 
and  in  the  organs  of  several  mammals. 

Finally,  the  phenomena  of  internal  destruction  which  we 
have  had  occasion  to  observe  in  yeasts,  can  be  attributed  to 
oxidizing  diastatic  actions. 

We  have  found  that  by  reducing  a  certain  quantity  of 
compressed  yeast  to  minute  fragments  and  then  heaping 
them  up,  an  increase  in  temperature  is  soon  manifest,  which 
may  reach  400  at  the  end  of  2  hours.  This  temperature  may, 
for  example,  be  obtained  with  2  kilograms  of  fresh  yeast 
ground  and  massed  in  heaps  20  centimetres  in  height  at  a 
temperature  of  200.  The  same  experiment,  made  in  vacuo, 
does  not  result  in  the  least  elevation  of  temperature.  The 
experiment  may  be  made  in  the  following  manner: 

In  a  half-litre  flask,  provided  with  three  tubes,  dispose 
layers  of  yeast  reduced  to  small  fragments  and  alternating 
with  layers  of  pumice-stone,  which  prevents  the  yeast  from 


OXIDASES.  295 

settling.  A  thermometer  is  introduced  in  the  center  tube 
and  a  current  of  air  established  with  the  other  two.  As  soon 
as  the  air  enters  the  flask  the  temperature  rises,  and  if  the 
tap  is  closed  to  the  air  it  is  immediately  found  to  decrease. 

By  allowing  the  experiment  to  continue  for  several  hours, 
it  can  be  renewed  several  times  with  the  same  yeast,  for  3  or 
4  consecutive  days  ;  and  it  will  be  observed  that  at  each  entry 
of  the  air  in  the  bottle  the  temperature  rises.* 

When,  on  the  contrary,  air  is  allowed  to  pass  into  the 
bottle  for  5  or  6  consecutive  hours,  the  yeast  liquefies  and  is 
completely  exhausted. 

By  crushing  the  yeast  with  pumice-stone  in  a  powerful 
crusher  a  paste  is  obtained  which,  allowed  to  stand  with  cold 
water  and  filtered,  gives  a  liquid  free  from  cells  and  yet  offer- 
ing, from  the  point  of  view  of  oxidation,  the  same  properties 
as  the  yeast  itself. 

The  fragments  of  pumice-stone,  impregnated  with  liquid, 
put  in  a  mass  of  glycogen  in  the  air  produce  there  an  eleva- 
tion of  temperature  of  from  4  to  6  degrees.  This  extract  is 
less  active  than  the  yeast  itself,  but  a  series  of  experiments 
have  shown  us  that  it  possesses,  like  yeast,  an  oxidizing  dia- 
static  power. 

In  view  of  all  these  facts  it  is  unquestionable  that  the 
phenomena  of  respiration  and  oxidation  of  vegetables  and 
animals  must  be  generally  attributed  to  oxidases. 

It  is  seen,  after  this  short  exposition,  that  the  discovery 
of  oxidases  was  of  considerable  importance,  because  it  has 
thrown  some  light  on  phenomena  still  unexplained,  or  which 
were  explained  by  erroneous  theories. 

The  study  of  oxidizing  enzymes  has  also  much  interest 
from  a  chemical  point  of  view  for  they  constitute  very  sensi- 
tive reagents  for  many  organic  substances. 


*  This  experiment  is  specially  interesting  from  the  point  of  view  of 
gas  analysis.  We  have,  in  fact,  observed  that  one  can  in  this  way  dis- 
tinguish 1  per  cent  of  oxygen  when  mixed  with  inactive  gases. 


296  THE  ENZYMES   AND    THEIR  APPLICATIONS. 

General  Properties  of  Oxidases- — Like  all  diastases,  oxi- 
dases are  extremely  unstable  bodies.  They  are  destroyed 
by  heat  above  6o°. 

Antiseptics,  in  general,  appear  capable  of  simply  retard- 
ing the  oxidation  produced  by  these  agents.  This  retarding 
action  of  antiseptics  has  not,  however,  been  generally  estab- 
lished. We  think,  on  the  contrary,  that  the  different  dia- 
stases belonging  to  this  class  are  more  or  less  sensitive  to  the 
action  of  antiseptics,  and  that  to  this  fact  must  be  attributed 
the  negative  results  of  a  number  of  investigations  carried  on 
with  bodies  which  certainly  contain  oxidases. 

Alcohol,  when  sufficiently  dilute,  does  not  appear  to  hin- 
der the  action  of  enzymes  of  this  class.  The  diastase  of 
latex,  laccase,  still  produces  an  oxidation  in  a  50  per  cent 
alcoholic  solution. 

Soluble  oxidizing  ferments  give  a  strong  blue  color  to 
tincture  of  guaiacum  to  which  hydrogen  peroxide  is  not 
added,  guaiaconic  acid  being  formed  with  the  oxygen  ab- 
sorbed from  the  air. 

Temperature,  as  well  as  the  reaction  of  the  medium,  in- 
fluences the  action  of  oxidases. 

Finally,  the  greater  number  of 'oxidases  act  especially  on 
bodies  of  the  aromatic  series :  phenols,  amines,  and  their  sub- 
stitution products. 

The  oxidation  products  brought  about  by  diastases  are 
as  yet  poorly  defined.  The  oxidation  of  bodies  of  the  aro- 
matic series  is  produced  either  by  an  elimination  of  the  hy- 
drogen or  by  direct  addition  of  oxygen.  This  oxidation  is 
never  complete.  The  oxidation  of  fatty  substances  is  much 
more  energetic ;  it  leads  to  a  complete  destruction  and  to  the 
formation  of  carbonic  acid. 

The  action  of  oxidases  is  not  at  all  specific.  Laccase,  for 
example,  transforms  hydroquinone  (diatomic  phenol)  just  as 
well  as  pyrogallol  (triatomic  phenol). 

The  position  of  the  groups  appears,  however,  to  play  a 


OXIDASES.  297 

principal  part ;  the  para  position,  for  example,  seems  to  in- 
fluence the  reaction  favorably. 

Among  the  diastases  producing  hydrolysis  the  individu- 
ality is  more  strongly  marked;  sucrase,  for  example,  can 
only  decompose  saccharose  and  is  incapable  of  acting  on  very 
closely  related  bodies  which  differ  only  by  their  molecular 
configuration. 

The  quantity  of  oxygen  absorbed  under  the  action  of 
oxidizing  enzymes  may  serve,  in  most  cases,  to  measure  the 
intensity  of  oxidation. 

Preparation  of  Oxidases. — Oxidases  are  extracted  from 
bodies  which  contain  them  by  the  methods  generally -used  for 
the  extraction  of  soluble  hydrolyzing  ferments. 

The  bodies  serving  in  the  preparation  are  ground  and 
then  extracted  in  the  presence  of  chloroform.  The  use  of 
the  latter  body  constitutes  a  danger,  however,  for  it  is  not 
known  whether  this  antiseptic,  which  leaves  most  hydrolyz- 
ing diastases  intact,  is  also  without  action  on  all  the  oxidases. 
It  is,  therefore,  to  be  recommended,  in  the  preparation  of 
oxidases,  to  make  two  triturations,  one  with  water  and 
chloroform,  the  other  with  water  containing  ether.  In  cer- 
tain cases  the  oxidases  will  be  found  in  the  water  and 
ether,  while  the  chloroform  infusion  will  not  contain  a  trace 
of  active  substances. 

The  infusion  is  then  precipitated  by  alcohol ;  the  precip- 
itate formed  is  redissolved  and  reprecipitated  several  times 
to  purify  it. 

The  method  of  extraction  with  glycerin  is  also  applicable 
to  the  preparation  of  oxidases. 

LACCASE. 

Laccase  is  a  soluble  ferment  producing  the  oxidation  of 
the  latex  of  the  lac-tree  and  transforming  it  into  a  very  beau- 
tiful varnish  which  the  Japanese,  the  inhabitants  of  Tong- 


298  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

"king,  and  the  Chinese  use  for  varnishing  their  furniture.  The 
latex  is  a  clear  liquid  presenting  the  appearance  and  the  con- 
sistency of  honey.  It  is  collected  in  eastern  Asia  by  making 
incisions  in  the  bark  of  certain  resinous  trees  of  the  order 
Anacardiaceas  (Rhus  vermicifera). 

The  odor  of  the  latex  is  very  slight  and  somewhat  re- 
sembles that  of  butyric  acid;  it  has  an  acid  reaction. 

The  latex  changes  with  extraordinary  rapidity.  Ex- 
posed to  oxygen  it  turns  brown  and  its  surface  becomes 
covered  with  a  resistant  film  of  a  beautiful  black  color  and 
absolutely  insoluble  in  ordinary  solvents.  In  vacuo,  change 
does  not  occur  and  the.  latex  can  be  kept  for  a  very  long 
time. 

The  first  data  on  laccase  were  obtained  by  the  Japanese 
chemist  Hikorokuro  Yoshida.  The  study  of  the  oxidation 
•of  the  latex  revealed  to  him  the  presence  of  a  body  which 
lie  called  uruschic  acid  (C14H1902),  a  body  which  by  oxida- 
tion changes  into  oxyuruschic  acid,  as  is  shown  by  the  fol- 
lowing equation: 

2C14H1902  +  30  =  2C14H1803  +  H20. 

Uruschic  acid.  Oxyuruschic  acid. 

Bertrand,  by  diluting  the  latex  in  a  great  quantity  of  al- 
cohol, discovered  in  it  two  products,  one  which  enters  into 
solution,  while  the  other  is  precipitated. 

This  precipitate,  separated  from  the  liquid,  is  a  sort  of 
;§um.  It  is  carefully  washed  with  alcohol,  taken  up  again 
with  distilled  water,  then  precipitated  again  with  10  volumes 
of  alcohol.  Then  it  can  be  collected  in  the  form  of  flakes 
and  dried  in  vacuo.  The  product  obtained  by  this  method 
resembles  ordinary  gums  and  is,  like  them,  transformed  by 
"hydration  into  a  mixture  of  galactose  and  arabinose. 

This  body  possesses  a  diastatic  power. 

The  alcoholic  solution,  after  the  gummy  precipitate  has 
been  removed,  is  quickly  distilled  in  vacuo.     The  residue  is 


OXIDASES.  299 

shaken  in  water,  then  in  ether;  the  water  retains  the  glucose, 
mineral  salts,  etc.,  and  the  ether  dissolves  the  resinous  ex- 
tract of  the  latex.  The  ether  is  then  decanted,  and  evapor- 
ated in  an  atmosphere  of  hydrogen. 

The  product  obtained  by  this  method  is  laccol;  it  is  an 
oily  liquid,  with  a  high  density,  not  dissolving  in  water  but 
entirely  soluble  in  alcohol,  ether,  chloroform,  and  benzol. 
The  manipulation  of  this  product  presents  certain  dangers : 
traces  of  laccol  may  act  in  an  injurious  way  on  the  skin.  In 
the  air,  it  turns  a  reddish-brown  color,  becomes  somewhat 
viscous,  and  is  finally  converted  into  resin. 

Oxidation,  favored  by  potash  and  soda,  is  produced  in  dif- 
ferent stages.  The  liquid  becomes  warm,  turns  green,  then 
inky  black,  and  absorbes  a  great  quantity  of  oxygen.  Lac- 
col gives  with  ferric  chloride  and  lead  acetate  reactions  much 
resembling  the  reactions  which  the  polyatomic  phenols  pro- 
duce with  the  same  agents.     . 

In  the  presence  of  lacca.se,  the  oxidation  of  laccol  is  much 
more  pronounced,  much  more  rapid,  and  finally  gives  a  black 
insoluble  substance  which  is  not  obtained  in  the  absence  of 
the  enzyme. 

Bertrand,  at  the  beginning  of  his  studies,  thought  that 
the  addition  of  oxygen  was  effected  by  simple  chemical  affin- 
ity, and  that  laccase  then  acted  on  the  oxidized  bodies  in  the 
manner  of  a  hydrating  agent. 

In  the  course  of  his  experiments,  the  French  chemist  suc- 
ceeded in  determining  the  true  mechanism  of  oxidation. 
He  observed  that  the  quantity  of  oxygen  absorbed  by  the 
laccol  in  contact  with  the  air  increases  with  the  amount  of 
laccase  used,  which  can  only  be  explained  by  a  direct  oxidiz- 
ing action  of  laccase. 

Conclusive  proofs  were  afterwards  furnished  by  Bertrand. 
He  caused  a  certain  quantity  of  laccase  to  act  on  bodies 
nearly  related  to  laccol,  principally  on  hydroquinone  and  py- 
rogallol,  and  found  that  in  the  presence  of  laccase  all  the 
polyatomic  phenols  absorbed  a  certain  quantity  of  oxygen, 


3°°  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

liberating  carbonic  acid.  In  the  absence  of  the  enzyme,  on 
the  contrary,  or  even  with  a  diastatic  solution  heated  to  ioo°, 
no  oxidation  occurred.  The  oxidizing  action  of  laccase  is 
therefore  well  demonstrated. 

Bertrand  then  discovered  a  very  sensitive  reaction  for 
discerning  the  presence  of  oxidases  in  plants.  He  found 
that  the  alcoholic  tincture  of  guaiacum  takes,  in  the  pres- 
ence of  laccase,  a  deep  blue  color  by  the  action  of  air  alone, 
while  to  obtain  the  same  result  with  hydrolyzing  diastases, 
hydrogen  peroxide  must  be  used. 

The  same  reaction  takes  place  also  when  cuttings  of  or- 
gans containing  an  oxidizing  diastase  are  treated  with  tinc- 
ture of  guaiacum. 

The  sensitiveness  of  this  reaction  allowed  Bertrand  to 
recognize  the  presence  of  laccase  in  a  great  number  of  vege- 
tables, and  to  evolve  the  hypothesis,  which  is  moreover 
quite  justifiable,  that  laccase  is  distributed  all  through  the 
vegetable  kingdom.  This  diastase  has  been  found  in  the 
following  list  of  plants: 

Beets,  carrots,  turnips  (roots),  dahlias  (roots,  tubers),, 
potatoes  (tubers),  asparagus  (yellow  stem),  lucerne,  clover, 
ray  grass  (stems  and  leaves),  Jerusalem  artichokes,  apples, 
pears,  chestnuts,  gardenias  (flowers),  lac-tree  (latex). 

For  the  extraction  of  laccase,  secreted  by  the  vegetables, 
we  have  just  named,  Bertrand  made  use  of  a  method  slightly 
different  from  that  used  for  the  latex.  The  juice  ex- 
tracted from  the  parenchymatous  organs  of  the  rhizomes, 
or  tubers  is  precipitated  immediately  after  its  extraction. 
As  to  the  liquid  extracted  from  the  green  parts  of  the  plant, 
chloroform  is  added  to  it  and  it  is  allowed  to  stand  at  the 
ordinary  temperature  for  24  hours;  then  a  coagulum  forms 
which  is  separated  from  the  rest  of  the  liquid,  and  in  the 
filtered  liquid  the  precipitation  by  alcohol  is  accomplished. 
This  precipitation  is  made  in  the  same  way  as  for  the  latex  o£ 
the  lac-tree. 


OXIDASES.  3QI 

Bertrand  observed  that  the  greatest  quantity  of  laccase 
is  secreted  by  the  organs  in  the  course  of  development. 

Emile  Bourquelot  and  Bertrand  sought  the  presence  of 
laccase  in  mushrooms,  plants  which,  as  we  know,  cause 
energetic  phenomena  of  oxidation. 

Schonbein,  in  1856,  had  already  made  the  curious  obser- 
vation, which,  moreover,  remained  as  a  simple  observation, 
that  the  juice  of  two  mushrooms,  Boletus  luridus  and  Aga- 
ricus  sanguineus,  colored  blue  tincture  of  guaiacum  with- 
out addition  of  hydrogen  peroxide  and  lost  this  faculty  when 
it  was  heated  to  ioo°. 

The  presence  of  oxidases  was  sought  for  by  the  French 
scientists  in  more  than  two  hundred  kinds  of  cryptogams  and 
the  reaction  of  guaiacum  was  tried  in  the  various  organs 
of  these  plants.  They  examined  especially  the  Basidio- 
mycetes,  some  Ascomycetes,  a  Myxomycete, — Reticularia 
maxima,  the  Polypori,  and  the  Agaricines.  Russula  f ceteris, 
Persoon,  was  studied  particularly  on  account  of  the  pecu- 
liarity which  all  its  parts  have  of  coloring  blue  with  a  solution 
of  guaiacum.  The  investigators  cut  and  crushed  125  grams 
of  Russula,  then  soaked  it  in  water  with  chloroform  added. 
The  filtered  liquid  took  in  the  course  of  an  hour  pale  yel- 
low, then  dirty  red  tints;  it  presented  all  the  characteristics 
of  a  solution  of  laccase. 

The  oxidizing  diastase  of  these  different  plants  is  soluble, 
at  least  in  part,  in  alcohol,  for  when  an  excess  of  this  reagent 
is  added  to  the  diastatic  solution,  even  when  the  latter  is  very 
active,  only  a  very  weak  precipitate  is  obtained. 

Below  is  the  table  which  Bourquelot  and  Bertrand  give 
as  a  summing  up  of  their  experiments,  from  which  it  is  seen 
that  the  oxidizing  enzyme  is  found  in  plants  destitute  of 
chlorophyll. 

In  the  mushrooms  it  is  distributed  throughout  the  whole 
reproductive  portion ;  it  is  found  localized  in  the  lamellae  of 
certain  hymenomycetes,  or  at  the  base  of  the  stipe. 


302  THE   ENZYMES  AND    THEIR   APPLICATIONS. 

„                                                xt       u         r                   Species. 
Genus  Number  of  \__^ 

sPecieS,  With  Withou? 

sub-genus.  examined.       laccasei         laccase- 

Russula 18  18  o 

Lactarius 20  18  2 

Psalliota 5  4  1 

Boletus 18  10  8 

Clitocybe 9  5  4 

Marasmius 6  o  6 

Hygrophorus 6  o  6 

Cortinarius 12  1  11 

Inocybe 6  1  5 

Amanita 7  2  5 

Manner  of  Action  of  Laccase. — Laccase  acts  on  a  large 
number  of  substances.  Added  to  a  solution  of  hydroquinone 
in  an  open  vessel,  it  produces  comparatively  rapid  oxida- 
tion. The  solution  takes  a  deep  color  and  at  the  end  of 
some  time  crystalline  plates  of  a  green  color  are  formed. 

The  oxidized  liquid  has  the  characteristic  odor  of 
quinone,  and  the  reaction  may  be  expressed  by  the  following 
equation: 

2C6H4<(g^  +  03=2C6H4<° 

Hydroquinone.  Quinone. 

The  diastase  also  acts  on  gallic  acid,  but  the  product 
of  the  reaction  has  so  far  been  little  studied. 

By  causing  a  certain  quantity  of  laccase  extracted  from 
Russula  to  react  on  gallic  acid,  Bourquelot  and  Bertrand  ob- 
tained the  following  results: 
Quantities  used: 

Gallic  acid 1  gr. 

Water 100  c.c. 

Laccase  solution 5  c.c. 

After  one  hour: 

Oxygen  absorbed 15.9  c.c. 

Carbonic  acid  freed 13.9  c.c. 


oxidases:  303 

After  four  hours: 

Oxygen  absorbed 17.6  c.c. 

Carbonic  acid  freed 1 1. 1  c.c. 

CO. 
After  an  hour  the  ratio    equals  0.874  and  after  four 

hours  0.630.     These  quite  high  ratios  show  that  the  oxidiz- 
ing power  of  laccase  is  very  great. 

By  trying  the  action  of  laccase  on  three  isomeric  poly- 
phenols :  on  hydroquinone,  pyrocatechin,  and  resorcin,  the 
following  figures  have  been  obtained  which  give  an  idea 
of  the  rapidity  of  the  oxidation: 

Oxygen  absorbed.  C02  freed. 

Hydroquinone  (para-diphenol).    After  4  h 32.0  c.c.  1.7  c.c. 

Pyrocatechin  (ortho-diphenol).  "      4  b. 17.4     "  2.8    " 

Resorcin  (meta-diphenol)  "    15  h 0.6     "  0.0    " 

It  is  seen  that  the  quantity  of  oxygen  absorbed  is  almost 
nought  for  the  meta-diphenol,  while  the  para-diphenol  oxi- 
dizes very  strongly. 

These  facts  are  reproduced  in  all  Bertrand's  experiments; 
phloroglucin,  where  all  the  hydroxyls  are  in  the  meta  po- 
sition, refuses,  so  to  speak,  all  oxidation,  while  its  isomer, 
pyrogallol,  absorbs  oxygen  with  rapidity. 

The  different  polyphenols  examined  by  Bertrand  have 
shown  that  their  oxidizability  is  in  direct  proportion  to  the 
facility  with  which  they  are  transformed  into  quinones. 

The  whole  or  a  part  of  the  hydroxyls  of  polyphenols  may 
be  replaced  by  amido  radicals  (NH2),  without  the  progress 
of  oxidation  being  modified. 

The  paramidophenol : 

CH<OH(i) 
6    4     NH2(4) 

is  easily  oxidized;  metamidophenol,  on  the  contrary, 

CH<OH(i) 

6    4     NH2(3) 

takes  up  only  the  smallest  quantities  of  oxygen. 

According  to  J.  de  Rey  Pailhade,  laccase  exists  in  germi- 
nating grains.     The  enzyme  acts  on  an  oxidizable  material, 


304  THE  ENZYMES   AND    THEIR   APPLICATIONS. 

philothion,  also  contained  in  these  grains.  Laccase  conse- 
quently would  play  a  part  in  the  respiration  of  vegetable 
cells.  Still,  he  has  not  shown  at  all  that  the  oxidizing  en- 
zyme found  in  the  grains  is  laccase.  It  is  quite  possible  to 
believe  that  it  is  some  other  oxidase. 

We  can  now  define  in  a  more  general  way  the  manner 
of  action  of  laccase. 

Laccase  is  a  soluble  ferment  producing  the  oxidation  of 
bodies  of  the  benzene  series,  which  possess  at  least  two 
groups,  OH  or  NH2  ,  when  these  groups  occupy  the  para- 
or  ortho  position. 

Here  the  observations  specially  applicable  to  laccase 
cease.  The  later  works  of  Bourquelot  and  Bertrand  relate 
to  another  diastase,  or  rather  to  a  mixture  of  laccase  and 
another  enzyme,  tyrosinase,  whose  presence  investigators 
have  recognized  in  a  great  number  of  vegetables. 

TYROSINASE. 

The  juices  extracted  from  beets  and  some  other  plants, 
when  put  in  contact  with  the  air,  become  red  in  color,  then 
black.  This  phenomenon  is  due  to  the  oxidation  of  the 
tyrosin  which  is  found  in  these  plants,  and  which  is  caused 
by  the  action  of  a  diastase. 

The  rational  formula  of  tyrosin  or  oxyphenyl-amidopro- 
pionic  acid  is : 

rH    .COOH 
-       V2    3    NH, 


HC         CH 

(I  I 

HC         CH 


C 

I 
OH 


OXIDASES.  3°  5 

It  is  seen  by  this  formula  that  tyrosin  does  not  belong- 
wholly  to  the  class  of  bodies  we  have  recognized  as  being 
surely  oxidizable  by  laccase,  that  is,  to  the  class  of  poly- 
phenols whose  hydroxyls  are  in  the  para-  or  ortho-position. 

Tyrosin,  in  fact,  when  submitted  to  the  action  of  laccase 
does  not  absorb  oxygen. 

On  the  other  hand,  Bertrand  found  by  various  experi- 
ments that  the  oxidation  of  tyrosin  did  not  occur  when  the 
juice  extracted  from  the  plants  had  been  heated  to  ioo°. 
This  fact  indicated  the  intervention  of  a  diastase. 

The  oxidation  of  tyrosin  may  be  explained  by  the  action 
of  laccase  on  a  product  of  decomposition  of  tyrosin,  previ- 
ously elaborated  by  another  non-oxidizing  enzyme  contained 
in  the  liquid. 

To  test  this  hypothesis,  Bertrand  placed  in  a  flask  a  cer- 
tain amount  of  an  extract  of  Russula  and  a  few  grams  of 
tyrosin.  After  24  hours,  the  whole  was  heated  to  ioo°;  the 
addition  of  laccase  to  the  liquid  and  exposure  to  the  air  did 
not  then  cause  any  oxidation. 

It  was  therefore  demonstrated  by  this  experiment  that 
laccase  is  absolutely  without  action  on  tyrosin,  and  that  there 
is  not  produced  in  the  liquid  a  previous  diastatic  action  which 
permits  the  oxidation  by  laccase. 

In  reality,  the  absorption  of  oxygen  is  caused  by  the 
mediation  of  tyrosinase,  an  enzyme  similar  to  laccase  but 
acting  on  other  bodies. 

Tyrosinase  has  been  isolated  by  Bertrand  from  many 
plants.  Extracted  from  potatoes,  dahlias,  etc.,  it  possesses 
only  a  very  feeble  oxidizing  power;  but  extracted  from  fungi 
it  oxidizes  tyrosin  very  rapidly. 

Bourquelot  prepared  tyrosinase  with  Russula  nigricans, 
which  he  crushed  in  water  to  which  chloroform  had  been 
added.  The  liquid,  after  filtration,  constituted  the  diastatic 
solution. 

To  demonstrate  the  action  of  tyrosinase,  5  cubic  centi- 
metres of  the  diastatic  solution  are  put  in  reaction  tubes, 


306  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

then  5  cubic  centimetres  of  a  solution  of  tyrosin,  and  the 
mixture  is  shaken  from  time  to  time  to  introduce  the  air. 
The  liquid  becomes  first  red,  then  black. 

By  this  reaction  and  that  of  guaiacum,  Bourquelot  dem- 
onstrated the  existence  of  tyrosinase  in  the  following  fungi: 

Boletus,  Russula,  Lactarius,  Parillus,  Psalliota,  Hebeloma, 
Amanita,  Scleroderma. 

Certain  fungi  give  no  reaction,  either  with  guaiacum  or 
with  tyrosin,  and  it  may  be  concluded  from  this  that  they 
contain  no  oxidizing  enzyme. 

Tyrosinase  is  not  so  widely  distributed  in  nature  as  lac- 
case  ;  but  it  is  very  often  met  with,  simultaneously  with  the 
latter,  in  the  same  vegetable  juice.  Certain  diastatic  solu- 
tions extracted  from  plants  really  transform  tyrosin  as  well 
as  polyphenols. 

In  a  series  of  experiments  made  at  500,  6o°,  and  yoQ  Ber- 
trand  observed  that  the  faculty  which  vegetable  juices  pos- 
sess of  transforming  tyrosin  disappear  at  relatively  low  tem- 
peratures, while  the  similar  properties  of  laccase  still  persist 
in  the  liquid  at  higher  temperatures.  This  difference  in  re- 
sistance of  the  two  diastases  allows  the  separation  of  one 
from  the  other  in  the  following  manner: 

One  thousand  five  hundred  grams  of  fresh  Russula  delica 
are  reduced  to  a  pulp  and  macerated  in  an  equal  weight  of 
cold  water  to  which  chloroform  has  been  added.  By  pressing 
out  the  juice  of  the  paste  thus  obtained,  about  2  litres  of  a 
mucilaginous  liquid  is  obtained,  to  which  are  added  3  litres  of 
95  per  cent  alcohol ;  a  precipitate  is  formed  which  is  separated 
by  filtration.  The  alcoholic  liquid,  from  which  the  precip- 
itate has  been  separated,  is  reduced,  by  distillation  in  vacuo 
at  500,  to  about  half  a  litre.  The  product  thus  obtained  oxi- 
dizes hydroquinone  and  pyrogallol  very  rapidly,  leaving  the 
tyrosin  perfectly  intact.  The  precipitation  with  alcohol  and 
the  heating  to  500  have  destroyed  every  trace  of  tyrosinase. 

This  latter  diastase  is  found  in  the  precipitate  which  has 
been  separated  from  the  alcoholic  liquid.     This  precipitate  is 


OXIDASES.  307 

purified  by  diluting  with  water  containing  chloroform ;  it  is 
again  precipitated  with  2  volumes  of  alcohol  and  separated 
from  the  liquid.  The  product,  after  a  second  similar  treat- 
ment, is  dried  at  35 °.  It  reacts  with  difficulty  on  polyphenols, 
but  causes  a  very  rapid  oxidation  of  tyrosin. 

The  individuality  of  the  two  enzymes  is  then  well  proved. 

Influence  of  the  Medium  on  Oxidation.— Bourquelot,  in  a 
very  complete  work,  has  shown  the  relation  existing  be- 
tween the  composition  of  the  medium  and  the  diastatic  ac- 
tivity of  the  oxidizing  ferment  of  fungi,  a  ferment  composed, 
as  we  have  seen,  of  at  least  two  oxidizing  enzymes :  laccase 
and  tyrosinase. 

A  solution  of  anilin,  in  the  presence  of  an  infusion  of  fungi 
rich  in  oxidase,  oxidizes  very  slowly,  for  only  a  slight  change 
of  color  is  observed. 

Bourquelot  was  then  led  to  inquire  if  the  alkalinity  which 
anilin  gives  to  the  medium  did  not  exercise  an  unfavorable  in- 
fluence on  the  oxidizing  action  of  the  enzyme,  and  he  studied 
the  oxidation  of  anilin  in  the  presence  of  increasing  amounts 
of  acetic  acid. 

The  fungus  chosen  for  these  experiments  was  the  Russitla 
dclica,  because  the  filtered  juice  obtained  from  its  maceration 
gives  a  clear  aqueous  solution,  which  consequently  makes  it 
easy  to  observe  the  changes  in  color.  It  was  soaked  by  tak- 
ing 5  parts  of  water  for  one  part  of  fungus,  and  thus  was  ob- 
tained by  filtration  a  liquid  but  slightly  colored  yellow. 

This  extract,  with  the  addition  of  glacial  acetic  acid  in 
amounts  varying  from  1  to  50  parts  per  thousand,  was  tested 
with  tincture  of  guaiacum. 

Bourquelot  then  saw  the  blue  coloration  appear  with  the 
same  intensity  and  the  same  speed  in  all  the  experiments 
which  he  made.  Therefore,  the  reagent  is  not  influenced  by 
great  amounts  of  acetic  acid  and,  under  these  conditions,  the 
influence  of  the  acid  on  the  action  of  oxidase  may  be  studied. 
This  action  is  shown,  for  different  amounts  of  acid,  in  the  fol- 
lowing table: 


3°8 


THE  ENZYMES  AND    THEIR  APPLICATIONS. 


Control 
Test. 

Exper.  i. 

Exper.  a. 

Exper.  3. 

Exper.  4. 

Exper.  5. 

Exper.  6. 

Solution  of 

saturated 

aniline. 

5  c.c. 

5  c.c. 

5  c.c. 

5  c.c. 

5  c.c. 

5  c.c. 

5  c.c. 

Water. 

8  c.c. 

8  c.c. 

8  c.c. 

8  c.c. 

8  c.c. 

8  c.c. 

8  c.c. 

Acetic  acid,  % 

o 
5  c.c. 

0.1 

0.2 

0.4 

1 

2 

5 

Diastatic 
solution. 

5  c.c. 

5  c.c. 

5  c.c. 

5  c.c. 

5  c.c. 

5  c.c. 

Result. 

Slight 
oxida- 
tion. 

A  little 
stronger 
oxida- 
tion. 

Strong 
oxida- 
tion. 

Very 
strong 
oxida- 
tion. 

Strong 
oxida- 
tion. 

Very 
slight 
oxida- 
tion. 

No 
oxida- 
tion. 

Oxidation  hardly  appears  in  the  control  tube,  which  takes 
a  dirty  yellow  tint;  it  increases  with  extraordinary  rapidity 
in  experiments  1,  2,  3,  4,  where  the  solution  is  immediately 
colored  a  dirty  yellow,  with  the  formation  of  a  brownish-yel- 
low precipitate,  soluble  in  ether.  As  to  experiments  5  and  6 
containing,  respectively,  2  and  5  per  cent  of  acetic  acid,  the 
first  furnishes  a  slight  oxidation,  while  in  the  second  there  is 
absolutely  no  oxidation.  Therefore,  2  per  cent  acetic  acid 
is  unfavorable  to  oxidation. 

With  orthotoluidin  and  paratoluidin,  tried  under  the 
same  conditions,  with  the  same  quantities  of  acid,  the  same 
reactions  occur,  although  giving  different  colorations. 

Orthotoluidin  gives  a  transparent  violet  color,  becoming 
opaque  at  the  end  of  several  hours. 

An  aqueous  solution  of  phenol  takes  a  brown  tint  in  the 
presence  of  the  diastatic  solution.  This  reaction,  which  takes 
place  very  slowly,  is  wholly  prevented  by  acetic  acid  and 
favored  by  amounts  of  0.1  to  0.4  per  cent  of  carbonate  of 
sodium.  In  general,  the  oxidation  of  substances  of  basic 
nature  is  favored  by  the  acidity  of  the  medium,  while  sub- 
stances of  acid  nature  oxidize  more  readily  in  an  alkaline 
medium.  This  influence  of  the  medium  on  the  progress  of 
oxidation  is  very  considerable. 


OXIDASES.  309 

Action  of  Oxidase  on  Phenols  Insoluble  in  Water. — Bour- 
-quelot  first  occupied  himself  with  the  action  of  oxidase  on 
phenols  which  are  soluble  in  water.  He  then  turned  his  at- 
tention to  the  action  of  oxidase  on  phenols  insoluble  in  water 
but  soluble  in  ethyl  alcohol  or  methyl  alcohol.  He  previously 
assured  himself  that  the  alcohols  used  as  solvents  and  suit- 
ably diluted  produced  no  change  in  the  oxidase  and  that  the 
phenomenon  of  oxidation  occurred  there  in  the  same  way  as 
in  the  watery  solutions. 

Assured  of  this,  Bourquelot  made  various  experiments  on 
phenols  soluble  in  these  reagents.  The  results  of  his  re- 
searches is  here  given : 

The  action  of  oxidase  was  tried  on  three  solutions  of  dif- 
ferent xylenols  containing  0.50  gr.  of  xylenol,  100  grams  of 
absolute  alcohol,  and  50  cubic  centimetres  of  water. 

The  orthoxylenol  (1,  2,  4),  a  body  melting  at  550  to  6o°, 
produced  a  white  precipitate  which  then  became  salmon  pink 
and  soluble  in  ether. 

Metaxylenol  (1,  3,  4),  a  liquid  whose  alcoholic  solution 
becomes  green  under  the  action  of  ferric  chloride,  was  im- 
mediately oxidized  and  gave  a  white  precipitate  which  then 
became  dirty  pink,  and  is  soluble  in  ether. 

Paraxylenol,  melting  at  740  or  75 °,  was  made  slightly 
turbid,  and  gave  a  pure  rose-colored  precipitate  insoluble  in 
ether. 

Experiments  upon  the  oxidation  of  thymol  were  made  in 
a  solution  having  the  following  composition : 

Thymol 0.50  gr. 

Water 40  c.c. 

Alcohol 10  c.c. 

Solution  of  carbonate  of  sodium  (2%) .  .  5  c.c. 

Diastatic  solution 50  c.c. 

The  solution  absorbs  19  cubic  centimetres  of  oxygen  and 
a  white  precipitate  is  formed  in  the  liquid. 

Carvacrol,   tried  under  the  same  conditions,  gives  rise 


3io  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

little  by  little  to  a  turbidity,  then  to  a  white  precipitate,  ab- 
sorbing 27.5  c.c.  of  oxygen. 

"  BROWNING  "    OF    WINES. 

The  "  browning  "  of  wines  is  a  disease  characterized  by 
the  oxidation  of  the  coloring  matter  of  the  wine  and  the  pre- 
cipitation of  this  material,  while  the  entire  liquid  becomes 
yellow. 

In  1895,  Gairaud  recognized  that  this  phenomenon  was 
due  to  the  action  of  a  diastase,  yet  without  clearly  attributing 
it  to  an  oxidase. 

P.  Martinand,  in  a  work  published  later,  identified  the 
diastase  producing  oxidation  of  the  coloring  matter  of  wine 
with  the  laccase  recently  discovered  by  Bertrand.  This  iden- 
tification was  entirely  erroneous.  Indeed,  it  was  later  recog- 
nized that  the  oxidase  of  wine  transforms  polyphenols,  while 
laccase,  though  hastening  the  oxidation  of  browning  wines, 
is  incapable,  of  itself,  of  producing  it  in  wines. 

Cazeneuve,  having  added  to  wines  a  certain  quantity  of 
laccase,  observed  only  an  imperceptible  alteration,  although 
the  diastatic  solution  used  was  very  active  and  strongly 
colored  blue  the  tincture  of  guaiacum.  The  diastase  causing 
the  oxidation  of  the  coloring  matter  is  then  a  well-deter- 
mined enzyme.  Cazeneuve  gave  to  it  the  name  of  cenoxi- 
dase. 

Preparation  of  (Enoxidase. — Cazeneuve  observed  the 
phenomenon  of  oxidation  in  Beaujolais  wine,  which  was  very 
sensitive  to  the  action  of  the  air;  he  isolated  the  diastase 
from  it  by  the  following  process : 

The  wine  is  submitted  to  the  action  of  an  excess  of  alcohol, 
which  precipitates  a  substance  having  the  appearance  of  a 
gum.  This  precipitate  is  taken  up  again  with  distilled  water, 
in  which  it  dissolves,  giving  an  opaline,  uncolored  solution. 
The  liquid  obtained  is  again  precipitated ;  the  new  precipitate 
is  dried  in  vacuo  and  then  obtained  under  the  form  of  a  gum 
impregnated  with  oxidase. 


OXIDASES.  3 1  r 

Secretion  of  (Enoxidase. — The  various  reactions  which 
characterize  the  diastase  of  the  browning  of  wines  are  iden- 
tical with  those  of  all  oxidases.  Like  other  soluble  ferments 
they  color  blue  the  tincture  of  guaiacum. 

The  reaction  of  guaiacum  was  tried  by  Martinand  on  ripe 
grapes,  and  it  revealed  the  presence  of  oxidases.  With  the 
juice  of  the  grape  he  succeeded  in  transforming  hydro- 
quinone  and  pyrogallol. 

Ripe  grapes  secrete  a  greater  quantity  of  cenoxidase 
than  green  grapes,  and  raisins  are  completely  destitute  of  it. 
The  fermented  juices  of  pears,  plums,  and  apples  are 
richer  in  cenoxidase  than  wine. 

The  secretion  of  cenoxidase  was  attributed  by  Laborde 
to  the  presence,  at  the  root  of  the  vine,  of  the  mould  Botrytis 
cincrca  ("  sweet  rot  "). 

Measurement  and  Properties  of  (Enoxidase. — The  meas- 
urement of  oxidases  presents  great  difficulties.  In  fact  these 
enzymes  do  not  always  exercise  their  action  with  liberation 
of  carbonic  acid,  which  is  easy  to  measure ;  oxygen  is  some- 
times combined  with  hydrogen  to  form  water  or  is  directly 
combined  with  the  oxidizable  materials.  Under  these  con- 
ditions, the  analysis  of  the  products  of  oxidation  becomes 
very  difficult. 

Laborde  has  based  a  method  of  measurement  on  the 
coloration  which  a  diastatic  liquid  assumes  in  the  presence 
of  tincture  of  guaiacum.  He  takes  as  unit  the  coloration 
which  is  acquired  by  20  cubic  centimetres  of  alcoholic  solu- 
tion of  guaiacum  with  the  addition  of  0.5  gr.  of  iodine,  and  he 
compares  the  coloration  obtained  in  the  same  tincture  by 
oxidase  with  this  unit  in  a  Dubosc  colorimeter. 

(Enoxidase  oxidizes  the  coloring  matter  of  French  and 
Italian  wines;  Spanish  and  Turkish  wines  undergo  its  action 
with  more  difficulty. 

Cazeneuve  found  that  the  coloring  matter  of  wine  is  a 
phenol-like  body.     It  is  transformed  by  oxidation,  likewise 


312  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

the  ethers,  alcohols,  essences,  etc.,  to  which  is  due  the 
bouquet  of  the  wine. 

When  wine  is  shaken  with  ether,  it  yields  to  this  reagent 
a  substance  having  the  characteristics  of  a  tannin.  After 
oxidation  of  the  wine,  only  the  smallest  quantities  of  this 
substance  are  found,  often  indeed  not  a  trace  is  to  be  dis- 
covered. 

Now,  neutral  wine  after  having  been  treated  with  ether 
undergoes  no  alteration  under  the  action  of  oxidases. 

The  browning  of  wines,  according  to  this  experiment, 
therefore,  appears  to  be  due  to  the  oxidation  of  a  particular 
substance. 

CEnoxidase  is  weakened  in  proportion  as  it  acts,  for  the 
quantity  of  oxygen  absorbed  at  the  beginning  is  greater  than 
that  absorbed  at  the  end  of  oxidation. 

By  introducing  air  into  half  a  litre  of  wine,  Laborde  found 
that  absorption  occurred  during  the  first  eight  days  and  that 
at  the. end  of  this  time  a  sudden  check  occurred.  The  meas- 
urement of  the  gas  absorbed  gave  the  following  figures  for 
three  different  wines: 

Oxygen  absorbed    COs  set  free  „    f;    COa 
per  litre.  per  litre.  o 

ist  experiment.  .  .  50.8  c.c.  32.4  c.c.  0.63 
2nd  experiment. .  .  81.0  c.c.  38  c.c.  0.47 
3rd  experiment. .  .      110.2  c.c.       63.8  c.c.       0.58 

This  table  shows  that  there  is  not  only  oxidation  of  the 
coloring  matter,  but  combustion  of  this  matter  and  produc- 
tion of  carbonic  acid. 

Lagati  observed  that  by  the  addition  of  ferrous  salts  the 
wines  oxidize  just  as  under  the  action  of  a  diastase.  The  pre- 
cipitate which  he  thus  obtained  is  identical  with  the  precipi- 
tate of  browned  wines ;  it  is  not  produced  if  protected  from 
the  air,  nor  in  the  presence  of  sulphurous  anhydride. 

This  author  attributed  oxidation  to  the  action  of  ferrous 
salts  alone,  but  this  opinion  was  refuted  by  Laborde  in  a  con- 


OXIDASES.  313 

elusive  manner.  Indeed,  the  greatest  amount  of  iron  in  the 
ferrous  state  contained  in  a  wine  can  absorb  only  10  cubic 
centimetres  of  oxygen,  while  browning  wine  absorbs  as  much 
as  no  cubic  centimetres  per  litre.  Therefore,  besides  the 
action  of  the  ferrous  salt,  that  of  a  diastase  is  also  exercised. 

Action  of  Temperature. — According  to  Cazeneuve,  cen- 
oxidase  is  but  slightly  sensitive  to  low  temperatures:  at  o° 
and  even  below  oxidation  still  occurs.  At  65 °  the  diastase 
is  not  entirely  destroyed;  for  the  destruction  to  be  complete, 
the  temperature  must  be  raised  to  yo°-y2°.  Martinand  fixed 
the  temperature  of  destruction  at  720  for  4  minutes  or  at 
35°  for  an  hour. 

Bouffard  made  interesting  experiments  on  this  subject. 
In  3  tubes,  A,  B,  C,  he  put  in  A,  an  aqueous  solution  of  the 
enzyme;  in  B,  the  same  solution,  with  a  certain  quantity  of 
io°  alcohol  added;  in  C,  a  solution  of  the  same  diastase  with 
0.5  gr.  of  tartaric  acid  added.  The  temperature  of  destruc- 
tion was  determined  for  each  experiment  and  the  following 
results  obtained: 

Temperature 
of  destruction. 

Neutral  aqueous  solution 72-5° 

Solution  +  alcohol  at  io° 6o° 

Solution  +  tartaric  acid 52-5° 

It  is  seen  that  the  presence  of  alcohol  and  tartaric  acid 
lower  the  temperature  of  destruction.  When  20  per  cent  of 
alcohol  is  added,  the  temperature  of  destruction  is  lowered 
by  50  more.  At  6o°,  according  to  the  same  author,  the  ac- 
tivity lasts  for  2  minutes,  then  decreases  and  disappears  com- 
pletely at  the  end  of  20  minutes. 

Laborde  studied  the  action  of  temperature  in  an  acid 
diastatic  liquid  containing  5  parts  of  oxidase.  He  brought 
these  liquids  to  different  temperatures  and,  after  cooling,  he 
determined  the  quantity  of  active  substance  remaining. 
These  experiments  gave  the  following  figures : 


314  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

Oxidase. 

Temperature.  , * — % 

Active.  Destroyed. 

6o° 2.30  2.70 

65 i-5o  3-5 

70 O.9O  4. 1 

75 0.75  4.25 

80 0.45  4.55 

85 :........   o  5 

The  temperature  of  destruction  of  oenoxidase  is,  there- 
fore, situated  between  700  and  75  °,  but  the  activity  of  the 
enzyme  diminishes  considerably  at  6o°. 

Action  of  Chemical  Agents. — According-  to  Martinand, 
an  addition  of  acid  retards  oxidation  and  an  addition  of  alkali, 
on  the  contrary,  is  favorable  to  the  combination  of  oxygen. 

However,  when  the  wine  already  possesses  a  quite  large 
natural  acidity  of  itself,  oxidation  occurs,  even  without  the 
addition  of  diastase. 

Concentrated  alcohol  decomposes  the  diastase,  but  dilute 
alcohol  and  wine  containing  up  to  9  per  cent  leave  it  abso- 
lutely intact. 

Tricalcium  phosphate  and  tartaric  acid  are  without  action, 
either  accelerating  or  retarding,  on  oxidation.  Formol 
(formic  aldehyde)  is  also  without  action. 

Gallic,  pyrocatechuic,  and  salicylic  acids  hinder  oxidation. 

Sulphurous  acid,  in  an  amount  of  0.01  to  0.08  parts  per 
litre,  checks  the  action  of  oenoxidase  and  causes  its  destruc- 
tion. This  fact  was  demonstrated  by  Boufrard  and  Caze- 
neuve.  Cazeneuve,  by  adding  to  a  certain  quantity  of  wine 
•0.004  grams  of  sulphurous  acid,  precipitated  the  diastase  of 
this  wine  by  the  ordinary  methods,  washed  the  precipitate  in 
alcohol,  and  collected  it.  After  some  time  the  precipitate, 
redissolved  in  water,  no  longer  gave  coloration  with  tincture 
of  guaiacum.  The  sulphurous  acid,  therefore,  acted  directly 
•on  the  diastase. 

CEnoxidase  is  extremely  unstable.      In  the  air  it  is  rapidly 


OXIDASES.  3 1 5 

destroyed  by  absorption  of  oxygen.     By  exposing  a  solution 
of  oxidase  to  the  air,  Laborde  obtained  the  following  figures : 

~          .                                                               Oxidase 
Duration  „ 

of  aeration.  D         •    •  , 

Remaining.    Lost. 

2  days 3.5  2.0 

4     "     2.8         2.7 

6     "     2.4         3.1 

12       "       O.8  4.7 

It  will  be  noticed  that  the  destruction,  which  is  rapid  at 
the  beginning,  slackens  very  perceptibly  after  the  second 
day. 

Other  Oxidations  of  Wine. — According  to  Martinand, 
oxidase  plays  an  important  part  in  the  improvement  of  wines 
with  age.  He  was  able,  in  fact,  to  produce  artificially,  by  the 
addition  of  oxidase,  an  ageing  of  a  Burgundy  wine. 

The  wine,  with  oxidase  added,  and  exposed  to  the  air  for 
48  hours,  took  on  a  yellower  color  and  the  perfume  of  an  old 
wine.  The  coloration  of  this  wine  corresponds  to  red-violet 
354  of  the  Salleron  wine  colorimeter  before  oxidation ;  after 
being  exposed  to  the  air,  in  the  presence  of  oxidase,  the  tint 
corresponds  to  the  third  red  404. 

The  oxidation  of  the  sugar  and  tartaric  acid  of  the  wine 
must,  according  to  Martinand,  be  attributed  to  a  cause  of  the 
same  kind. 

A  special  action  of  oxidase  has  been  found  in  certain 
American  grapes. 

These  grapes  have  a  disagreeable  taste  which  is  lost  by 
aeration;  but  when  they  are  kept  at  a  temperature  of  ioo°, 
they  retain  the  special  flavor  which  disappears  by  the  ad- 
dition of  oxidizing  diastase. 

OXIDIN. 

Boutroux,  in  studying  the  cause  of  the  coloration  of 
brown  bread,  discovered  in  the  bran  an  active  substance  re- 
sembling laccase  which  he  called  oxidin. 

When  the  bran  is  left  to  soak  for  a  half-hour  with  its 


3i 6  THE  ENZYMES  AND    THEIR  APPLICATIONS. 

volume  of  water,  there  is  obtained  by  filtration  through  a. 
porcelain  filter  a  clear  light-colored  liquid,  which,  protected 
from  the  air,  keeps  without  changing  its  color. 

Put  in  contact  with  the  air,  this  liquid  takes  on  a  brown 
tint  which  deepens  with  age  and  finally  becomes  black.  This 
coloration  does  not  occur  in  an  infusion  brought  up  to  ioo°. 

Boutroux  succeeded  in  separating  from  the  infusion  the 
oxidizing  enzyme,  and  the  substance  which  undergoes  oxida- 
tion. By  adding  alcohol  to  the  filtered  infusion,  the  oxidase 
precipitates  without  carrying  with  it  the  oxidizable  sub- 
stance. 

In  this  way  two  solutions  may  be  obtained  which  sepa- 
rately do  not  change  color  in  the  air  and  which,  mixed,  grow 
brown  under  the  influence  of  oxygen. 

To  prepare  oxidin  the  bran  is  soaked  in  an  atmosphere  of 
carbonic  acid  gas,  and  filtered  under  the  same  conditions. 
To  the  filtered  liquid  is  added  3  volumes  of  95  per  cent 
alcohol  and  the  precipitate  is  washed  with  82  per  cent  alcohol 
on.  a  paper  filter.  The  filter  is  impregnated  with  an  amor- 
phous substance,  which  is  brown  and  difficult  to  detach.  The 
filter  is  cut  in  pieces  and  dried  in  a  vacuum.  This  paper,  im- 
pregnated with  active  substance,  acts  energetically  on  the 
sterilized  infusion  of  bran ;  it  also  oxidizes  hydroquinone  like 
laccase. 

Oxidin  is  also  precipitated  by  sodium  chloride.  An  in- 
fusion of  bran  saturated  with  this  salt  does  not  color  in  the 
air.  The  enzyme  is  evidently  precipitated,  but  the  precipitate 
is  not  active. 

Oxidin  plays  a  very  important  part  in  the  coloration  of 
brown  bread,  but  in  this  phenomenon  amylase  also  is  con- 
cerned. The  two  enzymes  contained  in  the  bran  act  in  dif- 
ferent ways. 

The  intervention  of  oxidase  is  manifested  during  the 
preparation  of  the  dough  and  in  the  first  stages  of  panary  fer- 
mentation. 

The  oxidizable  material  of  the  bran  is  at  this  point  trans- 


OXIDASES.  3 1 7 

formed  into  coloring  matter.  The  oxidation  which  oxidin 
produces  is  checked  by  the  acidity,  and  when  panary  fermen- 
tation has  become  more  active,  oxidin  ceases  to  act. 

The  color  of  the  dough  becomes  still  deeper  by  cooking. 
In  this  stage  of  the  work,  amylase  intervenes.  The  starch, 
which  is  in  suspension  in  the  dough  before  cooking,  is  par- 
tially liquefied  through  the  influence  of  the  amylase  of  the 
bran.  An  intimate  mixture  is  brought  about  between  the 
parts  not  yet  liquefied.  The  mass  changes  in  structure  and 
this  change  causes  coloration.  The  coloration  of  the  flour 
may  be  also  influenced  by  a  substance  found  in  the  germ  of 
the  wheat.  According  to  a  verbal  communication  made  to 
me  by  Albiana,  Jr.,  of  Barcelona,  who  is  very  expert  on  ques- 
tions of  milling,  the  flour  obtained  with  grain  deprived  of  the 
germ  is  white  and  unchangeable,  while  the  presence  of  the 
germ,  even  in  relatively  small  quantity,  furnishes  a  dough 
which  colors  very  rapidly.  It  is  probable  that  the  germ 
contains  an  oxidase  or  some  similar  diastase. 


OLEASE. 


Fresh  olives,  when  in  heaps,  easily  undergo  fermentation. 
One  finds  an  increase  in  temperature,  a  liberation  of  carbonic 
anhydride  with  formation  of  acetic  acid  and  other  fatty  acids. 
Talomei  showed  that  this  fermentation  was  caused  by  an 
enzyme  which  he  called  olease. 

This  agent  is  sometimes  found  in  olive  oil,  in  which  it 
causes  a  very  great  change.  Under  its  action,  the  oil  be- 
comes rancid,  on  account  of  the  formation  of  fatty  acids,  and 
discolors  on  account  of  the  precipitation  of  the  coloring  mat- 
ter.    This  discoloration  is  favored  by  light. 

Olease  is  isolated  from  the  oil  by  stirring  with  water. 
Thus  a  watery  solution  of  the  enzyme  is  obtained  and  the  oil 
remains  unchanged. 

The  optimum  temperature  for  the  action  of  olease  is  be- 
low 350.     The  acidity  of  the  medium  checks  the  diastatic  ac- 


318  THE  ENZYMES  AND    THEIR.  APPLICATIONS. 

tion  and  it  is  owing  to  this  circumstance  that  the  change  in 
the  oil  is  often  not  very  extensive,  the  fatty  acid  formed  hin- 
dering the  action  of  the  olease. 

BIBLIOGRAPHY. 

G.   Bertrand. — Sur  la  laccase  de  l'arbre  a  laque.     Comptes  Rendus,   ier 

semestre,  1894,  p.  1215. 
Sur  la  laccase  et  le   pouvoir  oxydant   de   cette  diastase.     Comptes 

Rendus,  ier  semestre,  1895,  p.  266. 
Sur  la   recherche  et  la   presence   de  la  laccase  dans  les  vegetaux.   , 

Comptes  Rendus,  2^  semestre,  1895,  p.  166. 
■ Sur  les  rapports  qui  existent  entre  la  constitution  chimique  des  com- 
poses  organiques   et  leur  oxydabilite   sous  l'influence  de  la   laccase. 

Comptes  Rendus,  ier  semestre,  1896,  p.  1132. 
Sur   une    nouvelle    oxydase    ou    ferment    soluble    oxydant    d'origine 

vegetale.     Comptes  Rendus,  ier  semestre,  1896,  p.  1215. 
-. Presence  simultanee  de  la  laccase  et  de  la  tyrosinase  dans  le  sue  de 

quelques  champignons.     Comptes  Rendus,  2«  semestre,  1896,  p.  463. 
Bouffard. — Observations  sur  quelques  proprietes  de  l'oxydase  des  vins. 

Comptes  Rendus,  ier  semestre,  1897,  p.  706. 
• Rappel  d'une  note  precedente.     Comptes   Rendus,    ier  semestre,  p. 

1053- 
Em.     Bourquelot    and    Bertrand. — La    laccase    dans    les    champignons. 

Comptes  Rendus,  2e  semestre,  1895,  p.  788. 
Em.  Bourquelot. — Influence  de  la  reaction  du  milieu  sur  l'activite  du  fer- 
ment oxydant  des  champignons.     Comptes  Rendus,  2e  semestre,  1896, 

p.  260. 
■ Action  du  ferment  soluble  oxydant  des  champignons  sur  les  phenols 

insolubles  dans  l'eau.     Comptes  Rendus,  2^  semestre,   1896,  p.  423. 
L.   Boutroux. — Le  pain.     Bailliere  et  fils,   Paris. 
Cazeneuve. — Sur  quelques  proprietes  du  ferment  soluble  oxydant  de  la 

casse  des  vins.     Comptes  Rendus,  ier  semestre,  1897,  p.  781. 
Sur   le    ferment    soluble    oxydant    de    la   casse    des    vins.     Comptes 

Rendus,  ier  semestre,   1897,  p.  406. 
Laborde. — Sur  l'absorption  de  l'oxygene  dans  la  casse  des  vins.     Comptes 

Rendus,  2e  semestre,  1897,  p.  248. 
Sur  l'oxydase  du  botrytis  cinerea.     Comptes   Rendus,   ier  semestre, 

1898,  p.  536. 

Sur  la  casse  des  vins.     Comptes  Rendus,  1896,  p.  1074. 

E.   Bourquelot  and  Bertrand. — Le  bleuissement  et  le  noircissement  des 

champignons.     Soc.  de  Biologie  de  Paris,   1895. 
Lagati. — Sur    la    casse    des    vins;    role    du    fer.     Comptes    Rendus,    ief 

semestre,  1897,  p.  1461. 
V.  Martinand. — Sur  l'oxydation  et  la  casse  des  vins.     Comptes  Rendus, 

ier  semestre,  1897,  p.  512- 


OXIDASES.  319 

Talomei. — Olease.  Atti.  Ace.  di  Lincei.  Rnd.  1896.  Berichte  der  deutsche 
chem.  Gesellschaft,  1896. 

J.  de  Rey  Pailhade. — Etude  sur  les  proprietes  chimiques  de  l'extrait  al- 
coolique  de  levure  de  biere;  formation  d'acide  carbonique  et  absorp- 
tion d'oxygene. 

J.  de  Rey  Pailhade. — Roles  respectifs  du  philothion  et  de  la  laccase  dans 
les  grains  en  germination.     Comptes  Rendus,  1895,  p.   1162. 

L.  Lindet.- — Sur  l'oxydation  des  tannins  des  pommes  a  cidre.  Bulletin  de 
la  Soc.  chim.,  Paris,  1895;  Comptes  Rendus,   1895. 

Hikorokuro  Yoshida. — Journal  of  the  Chem.  Society,  1883. 

J.  Eft'ront. — Action  de  l'oxygene  sur  les  levures  de  biere.  Comptes  Ren- 
dus, CXXVII,  p.  326,  1898. 

Martinand. — Action  de  l'air  sur  le  mout  de  raisin  et  sur  le  vin.  Comptes 
Rendus,  1895,  p.  502. 


INDEX. 


Amygdalin,  268. 
Amylase,  100. 

alteration  of,  103. 

analysis  of,  139. 

industrial  application  of,  147. 

influence  of  antiseptics  on,  117. 

influence  of  chemical   agents  on, 
112. 

influence  of  lactic  acid  on,  114. 

influence    of    sulphuric    acid    on, 
112. 

influence  of  temperature  on,  109. 

in  moulds,  102. 

in  saliva,  102. 

liquefying  power,  115. 

preparation  of,  102. 

properties  of,  106. 
Amylo-dextrins,  173. 

Bacillus  glutinis,  170. 
Bacillus  levans,  171. 
Bacillus  panificans,  170. 
Betulase,  273. 
Bread,  fermentation,  168. 
Brewing,  155. 

Caroubin,  256. 
Caroubinase,  256. 
Cerealin,  170. 
Cerealose,  220. 
Chinese  yeast,  223,  230. 

manufacture  of,  234. 
Choum-choum,  223. 
Classification,  48-49. 
Cytase,  148,  255. 

Deterioration,  129. 
Dextrins.  126. 
Dextrins  of  Duclaux,  126. 
Dextrin  syrup,  163. 
Diastase,  estimation,  143. 
Diastase  of  Reichler,  136. 


Diastase:    precipitation    by   tannin. 

Distillation:      preparation      of     the 

mash,  179. 
Distilleries,  East  Asian,  235. 

Emulsin,  268. 

in  fungi,  268. 
Enzymes: 

action  of  heat  on,  16. 

chemical  composition,  SI. 

definition,  5. 

heat  producers,  8. 

manner  of  action,  24. 

mechanical  precipitation,  13. 

nomenclature,  46. 

properties,  12,  13. 

reaction  with  guaiacum,  17. 

variability,  15. 
Erythrozyme,  273. 

Fermentation,  panary,  168. 
Floor  malting,  151. 

Galactose,  248. 
Glucase,  208. 
Glucosides: 

enzyme  action  on,  43,  44. 

ferments  of,  267. 
Glycerides,  ferments  of,  262. 
Guaiacum,  17. 

Infusion  process,  187. 
Inulase,  249. 
Inulin,  249. 
Invertin  (see  Sucrase). 

Japanese  yeast,  223. 

Keplin,  248. 
Koji,  223. 

preparation  of,  224. 

321 


322 


INDEX. 


Laccase,  297. 
Laccol,  299. 
Lactase,  248. 
Lactose,  248. 
Leaven,  171. 
Levulose,  250. 
Lipase,  262. 

Malt,  195. 

bacteria  in,  180. 

Pilsen  type,  153. 
Malt-sugar  syrup,  163. 
Maltase,  208. 

in  moulds,  212. 
Maltose,  manufacture  of,  161. 
Malting,  149. 
Malt,  Munich  type,  153. 
Malto-dextrins,  158. 
Migen,  223. 

'  Molasses,  fermentation  of,  88 
Monobutyrin,  265. 
Moto,  227. 
Myrosin,  272. 

Nefrozymase,  208. 

CEnoxidase,  310. 
Olease,  317. 
Oxidasis,  174,  292. 

preparation  of,  297. 

properties  of,  296. 
Oxidin,  315. 

Panary  fermentation,  168. 

bacteria  in,  170. 
Pancreato-lipase,  265. 
Pectase,  251. 
Pectin,  251. 
Pectinase,  251. 


Pectose,  251. 
Papain,  30. 
Pepsin,  30. 
Peptase,  148. 
Pneumatic  malting,  151. 
Ptyalin,  132. 

Rhamnase,  273. 

Saccharification: 

color  changes  in,  107. 

for  distillation,  179. 

influence  of  temperature  on,  no.. 
Sake,  223. 

manufacture  of,  229. 
Secretion  diastase,  134. 
Sero-lipase,  265. 

Starches,  action  of  amylase  on,  127, 
Steapsin,  262. 
Sucrase,  50. 

deterioration,  76. 

estimation,  71. 
Symbiosis,  249. 

Taka-Koji,  237. 
Translocation  diastase,  134. 
Trehalase,  246. 
Trehalose,  246. 
Tyrosinase,  304. 

Zymase,  276. 

conditions  of  action,  281. 

fermenting  power  of,  279. 

industrial  application,  289. 

preparation  of,  277. 
Zymogens,  24, 
Zymogenesis,  23. 
Zymolysis,  24. 


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7 


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3  00 

4  00 

1  50 

1  50 

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2  00 

1  25 

1  00 

1  50 

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2  00 

MacCord's  Kinematics ;  or,  Practical  Mechanism 8vo,  5  00 

Mechanical  Drawing 4to,  4  00 

Velocity  Diagrams 8vo,  1  50 

Mahan's  Industrial  Drawing.     (Thompson.) 8vo,  3  50 

Poole's  Calorific  Power  of  Fuels 8vo,  3  00 

Reid's  Course  in  Mechanical  Drawing 8vo,  2  00 

"       Text-book     of    Mechanical    Drawing    and    Elementary 

Machine  Design 8vo,  3  00 

Richards's  Compressed  Air 12mo,  1  50 

Robinson's  Principles  of  Mechanism 8vo,  3  00 

Smith's  Press-working  of  Metals 8vo,  3  00 

Thurston's   Treatise   on  Friction   and   Lost   Work   in   Machin- 
ery and  Mill  Work 8vo,  3  00 

Animal   as  a   Machine  and   Prime  Motor  and   the 

Laws  of  Energetics ; . . .  12mo,  1  00 

Warren's  Elements  of  Machine  Construction  and  Drawing.  .8vo,  7  50 
Weisbaeh's  Kinematics  and  the  Power  of  Transmission.     (Herr- 
mann—Klein. )    8vo,  5  00 

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mann—Klein. )    8vo,  5  00 

Hydraulics  and  Hydraulic  Motors.     (Du  Bois.)  .8vo,  5  00 

Wolff's  Windmill  as  a  Prime  Mover 8vo,  3  00 

Wood's  Turbines 8vo,  2  50 

MATERIALS  OF  ENGINEERING. 

Bovey's  Strength  of  Materials  and  Theory  of  Structures.  .8vo,  7  50 
Burr's  Elasticity  and  Resistance  of  the  Materials  of  Engineer- 
ing   8vo,  5  00 

Church's  Mechanics  of  Engineering 8vo,  6  00 

Johnson's  Materials  of  Construction Large  8vo,  6  00 

Keep's  Cast  Iron 8vo,  2  50 

Lanza's  Applied  Mechanics 8vo,  7  50 

Martens's  Handbook  on  Testing  Materials.     (Henning.) . . .  .8vo,  7  50 

Merriman'a  Text-book  on  the  Mechanics  of  Materials. ..  .8vo,  4  00 

"           Strength  of  Materials 12mo,  1  00 

Metcalf's  Steel.    A  Manual  for  Steel-users 12mo,  2  00 

Smith's  Wire:  Its  Use  and  Manufacture Small  4to,  3  00 

Thurston's  Materials  of  Engineering 3  vols.,  8vo,  8  00 

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and  their  Constituents 8vo,  2  50 

Thurston's  Text-book  of  the  Materials  of  Construction 8vo,  5  00 

Wood's  Treatise  on  the  Resistance  of   Materials   and   an   Ap- 
pendix on  the  Preservation  of  Timber 8vo,  2  00 

"       Elements  of  Analytical  Mechanics 8vo,  3  00 

STEAM  ENGINES  AND  BOILERS. 

Carnot's  Reflections  on  the  Motive  Power  of  Heat.     (Thurston.) 

12mo,  1  50 
Dawson's  "  Engineering  "  and  Electric  Traction  Pocket-book. 

16mo,  morocco,  4  00 

Ford's  Boiler  Making  for  Boiler  Makers 18mo.  1  00 

Goss's  Locomotive  Sparks 8vo,  2  00 

Hemenway's  Indicator  Practice  and  Steam-engine  Economy. 

12mo,  2  00 

Hutton's  Mechanical  Engineering  of  Power  Plants 8vo,  5  00 

"        Heat  and  Heat-engines 8vo,  5  00 

12 


Kent's  Steam-boiler  Economy 8vo 

Kneass's  Practice  and  Theory  of  the  Injector 8vo 

MacCord's    Slide-valves 8vo 

Meyer's   Modern   Locomotive  Construction 4to 

Peabody's  Manual  of  the  Steam-engine  Indicator 12mo 

"         Tables   of  the  Properties  of   Saturated   Steam   and 

Other  Vapors 8vo 

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Heat-engines 8vo 

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Peabody   and  Miller.     Steam-boilers 8vo 

Pray's  Twenty  Years  with  the  Indicator Large  8vo 

Pupin's  Thermodynamics   of  Reversible   Cycles    in    Gases   and 

Saturated    Vapors.     (Osterberg.) 12mo 

Reagan's  Locomotive   Mechanism   and   Engineering 12mo 

Rontgen's  Principles  of  Thermodynamics.  (Du  Bois.) .  . .  .8vo 
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Smart's  Handbook  of  Engineering  Laboratory  Practice.  .  12mo 

Snow's  Steam-boiler  Practice 8vo 

Spangler's    Valve-gears 8vo 

"  Notes  on  Thermodynamics 12mo 

Thurston's  Handy  Tables 8vo 

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Thurston's  Handbook  of  Engine  and  Boiler  Trials,  and  the  Use 

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4  00 

1  50 

2  00 
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MECHANICS  AND  MACHINERY. 

Barr's  Kinematics  of  Machinery 8vo, 

Bovey's  Strength  of  Materials  and  Theory  of  Structures.  .8vo, 

Chorda!. — Extracts  from  Letters 12mo, 

Church's  Mechanics  of  Engineering 8vo, 

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Cromwell's  Treatise  on  Toothed  Gearing 12mo, 

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Dana's  Text-book    of   Elementary   Mechanics   for    the    Use   of 

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World's  Columbian  Exposition  of  1893 4to,  half  mor., 

Dn  Bois's  Elementary  Principles  of  Mechanics: 

Vol.  I. — Kinematics 8vo, 

Vol.   TL— Statics 8vo, 

Vol.  III.— Kinetics 8vo, 

13 


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4 

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3 

50 

Du  Bois's  Mechanics  of  Engineering.     Vol.  I Small  4to,  7  50 

Vol.11 Small  4to,  10  00 

Durley's  Elementary  Text-book  of  the  Kinematics  of  Machines. 

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Mather's  Dynamometers,  and  the  Measurement  of  Power.  12mo,  3  00 

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Kerr's  Power  and  Power  Transmission 8vo,  2  00 

Lanza's  Applied  Mechanics 8vo,  7  50 

MacCord's  Kinematics ;  or,  Practical  Mechanism 8vo,  5  00 

"          Velocity  Diagrams 8vo,  1  50 

Merriman's  Text-book  on  the  Mechanics  of  Materials 8vo,  4  00 

*  Miehie's  Elements  of  Analytical  Mechanics 8vo,  4  00 

Reagan's  Locomotive  Mechanism  and  Engineering 12mo,  2  00 

Reid's  Course  in  Mechanical  Drawing 8vo,  2  00 

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Richards's  Compressed  Air 12mo,  1  50 

Robinson's  Principles  of  Mechanism 8vo,  3  00 

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Smith's  Press- working  of  Metals 8vo,  3  00 

Thurston's   Treatise   on  Friction  and  Lost  Work   in  Machin- 
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"  Animal  as  a  Machine  and  Prime  Motor,  and  the 

Laws  of  Energetics 12mo,  1  00 

Warren's  Elements  of  Machine  Construction  and  Drawing.  .8vo,  7  50 
Weisbach's     Kinematics     and     the     Power    of    Transmission. 

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(man — Klein.)    8vo,  5  00 

Wood's  Elements  of  Analytical  Mechanics 8vo,  3  00 

"       Principles  of  Elementary  Mechanics 12mo,  1  25 

"       Turbines   8vo,  2  50 

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METALLURGY. 

Egleston's  Metallurgy  of  Silver,  Gold,  and  Mercury: 

Vol.  I.-Silver 8vo,  7  50 

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Keep's  Cast  Iron 8vo,  2  50 

Kunhardt's  Practice  of  Ore  Dressing  in  Lurope 8vo,  1  50 

Le  Chatelier's  High-temperature  Measurements.     (Boudouard— 

Burgess.)  12mo,  3  00 

Metcalf's  Steel.    A  Manual  for  Steel-users 12mo,  2  00 

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14 


MINERALOGY. 

Barringer's    Description    of    Minerals    of    Commercial    Value. 

Oblong,  morocco,  2  50 

Boyd's  Resources   of    Southwest   Virginia 8vo,  3  00 

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Cloth,  1  25 

"         Dictionary  of  the  Names  of  Minerals 8vo,  3  50 

Dana's  System  of  Mineralogy Large  8vo,  half  leather,  12  50 

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"      Minerals  and  How  to  Study  Them 12mo,  1  50 

"      Catalogue  of  American  Localities  of  Minerals .  Large  8vo,  1  00 

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Egleston's  Catalogue  of  Minerals  and  Synonyms 8vo,  2  50 

Hussak's     The     Determination     of     Rock-forming     Minerals. 

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•  Penfield's  Notes  on  Determinative  Mineralogy  and  Record  of 

Mineral  Tests 8vo,  paper,  50 

Rosenbusch's  Microscopical  Physiography  of  the  Rock-making 

Minerals.      (Idding's.) 8vo,  5  00 

•Tillman's  Text-book  of  Important  Minerals  and  Rocks.. 8vo,  2  00 

Williams's  Manual  of  Lithology 8vo,  3  00 


MINING. 

Beard's  Ventilation  of  Mines 12mo,  2  50 

Boyd's  Resources  of  Southwest  Virginia 8vo,  3  00 

"       Map  of  Southwest  Virginia Pocket-book  form,  2  00 

*  Drinker's     Tunneling,     Explosive     Compounds,     and     Rock 

Drills 4to,  half  morocco,  25  00 

Eissler's  Modern  High  Explosives 8vo,  4  00 

Goodyear's  Coal-mines  of  the  Western   Coast  of   the   United 

States    12mo,  2  50 

Ihlseng's  Manual  of  Mining 8vo,  4  00 

Kunhardt's  Practice  of  Ore  Dressing  in  Europe 8vo,  1  50 

O'Driscoll's  Notes  on  the  Treatment  of  Gold  Ores 8vo,  2  00 

Sawyer's  Accidents  in  Mines 8vo,  7  00 

Walke's  Lectures  on  Explosives 8vo,  4  00 

Wilson's  Cyanide  Processes 12mo,  1  50 

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Wilson's  Treatise  on  Practical  and  Theoretical  Mine  Ventila- 
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SANITARY  SCIENCE. 

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8vo,  3  00 

Water-supply    Engineering 8vo,  4  00 

Fuertes's  Water  and  Public  Health 12mo,  1  50 

Water-filtration    Works 12mo,  2  50 

15 


Gerhard's  Guide  to  Sanitary  House-inspection 16mo,  »1  00 

Goodrich's  Economical  Disposal  of  Towns'  Refuse . . .  Demy  8vo,  3  50 

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Kiersted's  Sewage  Disposal 12mo,  1  25 

Leach's    The   Inspection    and   Analysis    of   Food   with    Special 

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"        Examination    of    Water.      (Chemical    and    Bacterio- 
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Merriman's  Elements  of  Sanitary  Engineering 8vo,  2  00 

Nichols's  Water-supply.     (Considered  Mainly  from  a  Chemical 

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Ogden's  Sewer  Design 12mo,  2  00 

*  Price's  Handbook  on  Sanitation 12mo,  1  50 

Richards's  Cost  of  Food.    A  Study  in  Dietaries 12mo,  1  00 

Richards  and  Woodman's  Air,  Water,  and  Food  from  a  Sani- 
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Richards's  Cost  of  Living  as  Modified  by  Sanitary  Science.  12mo,  1  00 

*  Richards  and  Williams's  The  Dietary  Computer 8vo,  1  50 

Rideal's  Sewage  and  Bacterial  Purification  of  Sewage 8vo,  3  50 

Turneaure  and  Russell's  Public  Water-supplies 8vo,  5  00 

Whipple's  Microscopy  of  Drinking-water 8vo,  3  50 

Woodhull's  Notes  on  Military  Hygiene 16mo,  1  50 


MISCELLANEOUS. 

Barker's  Deep-sea  Soundings 8vo,  2  00 

Emmons's  Geological  Guide-book  of  the  Rocky  Mountain  Ex- 
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Large  8vo,  1  50 

Ferrel's  Popular  Treatise  on  the  Winds c 8vo,  4  00 

Haines's  American  Railway  Management 12mo,  2  50 

Mott's  Composition,  Digestibility,  and  Nutritive  Value  of  Food. 

Mounted  chart,  1  25 

"       Fallacy  of  the  Present  Theory  of  Sound 16mo,  1  00 

Ricketts's  History  of   Rensselaer  Polytechnic  Institute,   1824- 

1894 '. Small    8vo,  3  00 

Rotherham's  Emphasised  New  Testament Large  8vo,  2  00 

"  Critical  Emphasised  New  Testament 12mo,  1  50 

Steel's  Treatise  on  the  Diseases  of  the  Dog 8vo,  3  50 

Totten's  Important  Question  in  Metrology 8vo,  2  50 

The  World's  Columbian  Exposition   of  1893 4to,  1  00 

Worcester  and  Atkinson.     Small  Hospitals,  Establishment  and 
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Green's  Grammar  of  the  Hebrew  Language 8vo,  3  00 

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"       Hebrew  Chrestomathy 8vo,  2  00 

Gesenius's  Hebrew  and  Chaldee  Lexicon  to  the  Old  Testament 

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Letteris's  Hebrew  Bible 8vo,  2  25 

16 


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